CD137 binding fibronectin type III domains

ABSTRACT

FN3 domains that specifically bind to CD137, their conjugates, isolated nucleotides encoding the molecules, vectors, host cells, and methods of making and using them are useful in therapeutic and diagnostic applications.

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional Application Ser. No. 62/434,064, filed 14 Dec. 2016. The entire contents of the aforementioned application are incorporated herein by reference in their entireties.

FIELD OF THE INVENTION

The present invention relates to fibronectin type III domains that specifically bind to cluster of differentiation 137 (CD137) and methods of making and using the molecules.

BACKGROUND OF THE INVENTION

Advances in understanding of the requirements for tumor antigen recognition and immune effector function indicate that a potential strategy to enhance an anti-tumor immune response is to provide co-stimulation through an auxiliary molecule. The current model for T-cell activation postulates that naive T-cells require two signals for full activation: (i) a signal provided through the binding of processed antigens presented to the T-cell receptor by major histocompatibility complex (MHC) class I molecules; and (ii) an additional signal provided by the interaction of co-stimulatory molecules on the surface of T-cells and their ligands on antigen presenting cells.

CD137 (4-1BB) is a member of the TNF receptor superfamily and is an activation-induced T-cell costimulatory molecule. The receptor was initially described in mice (B. Kwon et al., P.N.A.S. USA, 86:1963-7 (1989)), and later identified in humans (M. Alderson et al., Eur. J. Immunol., 24: 2219-27 (1994); Z. Zhou et al., Immunol. Lett., 45:67 (1995)). The interaction of CD137 and the CD137 ligand (4-1BBL) activates an important costimulatory pathway. Signaling via CD137 upregulates survival genes, enhances cell division, induces cytokine production, and prevents activation-induced cell death in T cells. The importance of the CD137 pathway has been underscored in a number of diseases, including cancer (see, e.g., U.S. Pat. No. 7,288,638).

Expression of CD137 has been shown to be predominantly on cells of lymphoid lineage such as activated T-cells, activated Natural Killer (NK) cells, NKT-cells, CD4CD25 regulatory T-cells, and also on activated thymocytes, and intraepithelial lymphocytes. In addition, CD137 has also been shown to be expressed on cells of myeloid origin like dendritic cells, monocytes, neutrophils, and eosinophils. Even though CD137 expression is mainly restricted to immune/inflammatory cells, there have been reports describing its expression on endothelial cells associated with a small number of tissues from inflammatory sites and tumors.

The physiological events observed following CD137 stimulation on T-cells are mediated by NF-κB and PI3K/ERK1/2 signals with separate physiological functions. NF-κB signals trigger expression of Bcl-XL, an anti-apopotic molecule, thus resulting in increased survival, whereas PI3K and ERK1/2 signals are specifically responsible for CD137-mediated cell cycle progression (H. Lee et al., J. Immunol., 169(9):4882-8 (2002)). The effect of CD137 activation on the inhibition of activation-induced cell death was shown in vitro by Hurtado et al. (J. Hurtado et al., J. Immunol., 158(6):2600-9 (1997)), and in an in vivo system in which anti-CD137 monoclonal antibodies (mabs) were shown to produce long-term survival of superantigen-activated CD8+ T-cells by preventing clonal deletion (C. Takahashi et al., J. Immunol., 162:5037 (1999)). Later, two reports demonstrated, under different experimental conditions, that the CD137 signal regulated both clonal expansion and survival of CD8+ T-cells (D. Cooper et al., Eur. J. Immunol., 32(2):521-9 (2002); M. Maus et al., Nat. Biotechnol., 20:143 (2002)).

Altogether, CD137 stimulation results in enhanced expansion, survival, and effector functions of newly primed CD8+ T-cells, acting, in part, directly on these cells. Both CD4+ and CD8+ T-cells have been shown to respond to CD137 stimulation, however, it appears that enhancement of T-cell function is greater in CD8+ cells ((W. Shuford et al., J. Exp. Med., 186(1):47-55 (1997); I. Gramaglia et al., Eur. J. Immunol., 30(2):392-402 (2000); C. Takahashi et al., J. Immunol., 162:5037 (1999)). Based on the critical role of CD137 stimulation in CD8+ T-cell function and survival, agonism of the CD137/CD137L system provides a plausible approach for the treatment of tumors and viral pathogens.

Alternatively, while it has been shown that agonistic antibodies to CD137 and the ligand to CD137 enhance lymphocyte activation, the CD137 protein has the opposite effect. It inhibits proliferation of activated T lymphocytes and induces programmed cell death. These T cell-inhibitory activities of CD137 require immobilisation of the protein, arguing for transmission of a signal through the ligand/coreceptor (Schwarz et al., Blood 87, 2839-2845 (1996); Michel et al., Immunology 98, 42-46 (1999)).

The known human CD137 ligand is expressed constitutively by monocytes and its expression is inducible in T lymphocytes (Alderson et al., Eur. J. Immunol. 24, 2219-2227 (1994)). Monocytes are activated by immobilised CD137 protein and their survival is profoundly prolonged by CD137. (Langstein et al., J. Immunol. 160, 2488-2494 (1998); Langstein et al., J. Leuk. Biol. 65, 829-833 (1999)). CD137 also induces proliferation in peripheral monocytes (Langstein et al., 1999b). Macrophage colony-stimulating factor (M-CSF) is essential for the proliferative and survival-enhancing activities of CD137 (Langstein et al., J. Leuk. Biol. 65, 829-833 (1999); Langstein et al., Blood 94, 3161-3168 (1999)).

Signalling through CD137 ligand has also been demonstrated in B cells where it enhances proliferation and immunoglobulin synthesis. This occurs at interactions of B cells with CD137-expressing T cells or follicular dendritic cells (Pauly et al., J. Leuk. Biol. 72, 35-42 (2002)). It was postulated that similarly to the CD40 receptor/ligand system, which mediates T cell help to B cells after first antigen encounter, the CD137 receptor/ligand system may mediate co-stimulation of B cells by FDC during affinity maturation (Pauly et al., J. Leuk. Biol. 72, 35-42 (2002)).

Furthermore, soluble forms of CD137 are generated by differential splicing and are selectively expressed by activated T cells (Michel et al., Eur. J. Immunol. 28, 290-295 (1998)). Soluble CD137 is antagonistic to membrane-bound or immobilised CD137, and levels of soluble CD137 correlate with activation induced cell death in T cells (DeBenedette et al., J. Exp. Med. 181, 985-992 (1995); Hurtado et al., J. Immunol. 155, 3360-3367 (1995); Michel et al., Cytokine 12, 742-746 (2000)).

Thus, considering the complicated picture for CD137 involvement in divergent mechanisms of action and different cell types, there exists a need for reagents to accurately detect CD137 in tumor tissues and other samples and for new therapeutics that modulate the interaction between CD137 and the 4-1BBL ligand or that modulate the interaction between CD137 and other cellular targets.

SUMMARY OF THE INVENTION

The invention provides an isolated FN3 domain that specifically binds to CD137 protein.

The invention also provides an isolated FN3 domain that specifically binds to CD137 protein comprising the amino acid sequence of SEQ ID NOs: 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222, 223, or 224.

The invention also provides an isolated polynucleotide encoding the FN3 domain that specifically binds to CD137 protein.

The invention also provides a vector comprising the polynucleotide.

The invention also provides a host cell comprising the vector.

The invention also provides a method of producing the FN3 domain that specifically binds to CD137 protein, comprising culturing the isolated host cell under conditions that the FN3 domain that specifically binds to CD137 protein is expressed, and purifying the FN3 domain that specifically binds to CD137 protein.

The invention also provides a pharmaceutical composition comprising the FN3 domain that specifically binds to CD137 protein and a pharmaceutically acceptable carrier.

The invention also provides an anti-idiotypic antibody that specifically binds the FN3 domain that specifically binds to CD137 protein.

The invention also provides a kit comprising the FN3 domain.

The invention also provides a method of detecting CD137-expressing cancer cells in a tumor tissue, comprising

-   -   obtaining a sample of the tumor tissue from a subject; and     -   detecting whether CD137 protein is expressed in the tumor tissue         by contacting     -   the sample of the tumor tissue with the FN3 domain that         specifically binds CD137 protein comprising the amino acid         sequence of one of SEQ ID NOs: 45-224 and detecting the binding         between CD137 protein and the FN3 domain.         The invention also provides a method of isolating CD137         expressing cells, comprising obtaining a sample from a subject;     -   contacting the sample with the FN3 domain that specifically         binds to CD137 protein comprising the amino acid sequence of one         of SEQ ID NOs: 45-224, and     -   isolating the cells bound to the FN3 domains.         The invention also provides a method of detecting         CD137-expressing cancer cells in a tumor tissue, comprising     -   conjugating the FN3 domain that specifically binds to CD137         protein comprising the amino acid sequence of one of SEQ ID NOs:         45-224 to a detectable label to form a conjugate;     -   administering the conjugate to a subject; and     -   visualizing the CD137 expressing cancer cells to which the         conjugate is bound.

DETAILED DESCRIPTION OF THE INVENTION

As used in this specification and the appended claims, the singular forms “a,” “an,” and “the” include plural referents unless the content clearly dictates otherwise. Thus, for example, reference to “a cell” includes a combination of two or more cells, and the like.

“Fibronectin type III (FN3) domain” (FN3 domain) refers to a domain occurring frequently in proteins including fibronectins, tenascin, intracellular cytoskeletal proteins, cytokine receptors and prokaryotic enzymes (Bork and Doolittle, Proc Nat Acad Sci USA 89:8990-8994, 1992; Meinke et al., J Bacteriol 175:1910-1918, 1993; Watanabe et al., J Biol Chem 265:15659-15665, 1990). Exemplary FN3 domains are the 15 different FN3 domains present in human tenascin C, the 15 different FN3 domains present in human fibronectin (FN), and non-natural synthetic FN3 domains as described for example in U.S. Pat. No. 8,278,419. Individual FN3 domains are referred to by domain number and protein name, e.g., the 3rd FN3 domain of tenascin (TN3), or the 10^(th) FN3 domain of fibronectin (FN10).

“Centyrin” refers to a FN3 domain that is based on the consensus sequence of the 15 different FN3 domains present in human tenascin C.

The term “capture agent” refers to substances that bind to a particular type of cells and enable the isolation of that cell from other cells. Exemplary capture agents are magnetic beads, ferrofluids, encapsulating reagents, molecules that bind the particular cell type and the like.

“Sample” refers to a collection of similar fluids, cells, or tissues isolated from a subject, as well as fluids, cells, or tissues present within a subject. Exemplary samples are tissue biopsies, fine needle aspirations, surgically resected tissue, organ cultures, cell cultures and biological fluids such as blood, serum and serosal fluids, plasma, lymph, urine, saliva, cystic fluid, tear drops, feces, sputum, mucosal secretions of the secretory tissues and organs, vaginal secretions, ascites fluids, fluids of the pleural, pericardial, peritoneal, abdominal and other body cavities, fluids collected by bronchial lavage, synovial fluid, liquid solutions contacted with a subject or biological source, for example, cell and organ culture medium including cell or organ conditioned medium and lavage fluids and the like.

“Substituting” or “substituted” or “mutating” or “mutated” refers to altering, deleting of inserting one or more amino acids or nucleotides in a polypeptide or polynucleotide sequence to generate a variant of that sequence.

“Variant” refers to a polypeptide or a polynucleotide that differs from a reference polypeptide or a reference polynucleotide by one or more modifications for example, substitutions, insertions or deletions.

“Specifically binds” or “specific binding” refers to the ability of the FN3 domain of the invention to bind CD137 with a dissociation constant (K_(D)) of about 1×10⁻⁶ M or less, for example about 1×10⁻⁷ M or less, about 1×10⁻⁸ M or less, about 1×10⁻⁹M or less, about 1×10⁻¹⁰ M or less, about 1×10⁻¹¹ M or less, about 1×10⁻¹² M or less, or about 1×10⁻¹³ M or less. Alternatively, “specific binding” refers to the ability of the FN3 domain of the invention to bind CD137 at least 5-fold above the negative control in standard ELISA assay. The isolated FN3 domain of the invention that specifically binds CD137 may, however, have cross-reactivity to other related antigens, for example to the same predetermined antigen from other species (homologs), such as Macaca Fascicularis (cynomolgous monkey, cyno) or Pan troglodytes (chimpanzee).

“Library” refers to a collection of variants. The library may be composed of polypeptide or polynucleotide variants.

“Stability” refers to the ability of a molecule to maintain a folded state under physiological conditions such that it retains at least one of its normal functional activities, for example, binding to a predetermined antigen such as CD137.

“CD137” refers to human CD137 protein having the amino acid sequence of SEQ ID NO:44.

“Tencon” refers to the synthetic fibronectin type III (FN3) domain having the sequence shown in SEQ ID NO:1 and described in U.S. Pat. Publ. No. 2010/0216708.

A “cancer cell” or a “tumor cell” refers to a cancerous, pre-cancerous or transformed cell, either in vivo, ex vivo, and in tissue culture, that has spontaneous or induced phenotypic changes that do not necessarily involve the uptake of new genetic material. Although transformation can arise from infection with a transforming virus and incorporation of new genomic nucleic acid, or uptake of exogenous nucleic acid, it can also arise spontaneously or following exposure to a carcinogen, thereby mutating an endogenous gene. Transformation/cancer is exemplified by, e.g., morphological changes, immortalization of cells, aberrant growth control, foci formation, proliferation, malignancy, tumor specific markers levels, invasiveness, tumor growth or suppression in suitable animal hosts such as nude mice, and the like, in vitro, in vivo, and ex vivo (Freshney, Culture of Animal Cells: A Manual of Basic Technique (3rd ed. 1994)).

“Vector” refers to a polynucleotide capable of being duplicated within a biological system or that can be moved between such systems. Vector polynucleotides typically contain elements, such as origins of replication, polyadenylation signal or selection markers that function to facilitate the duplication or maintenance of these polynucleotides in a biological system. Examples of such biological systems may include a cell, virus, animal, plant, and reconstituted biological systems utilizing biological components capable of duplicating a vector. The polynucleotide comprising a vector may be DNA or RNA molecules or a hybrid of these.

“Expression vector” refers to a vector that can be utilized in a biological system or in a reconstituted biological system to direct the translation of a polypeptide encoded by a polynucleotide sequence present in the expression vector.

“Polynucleotide” refers to a synthetic molecule comprising a chain of nucleotides covalently linked by a sugar-phosphate backbone or other equivalent covalent chemistry. cDNA is a typical example of a polynucleotide.

“Polypeptide” or “protein” refers to a molecule that comprises at least two amino acid residues linked by a peptide bond to form a polypeptide. Small polypeptides of less than about 50 amino acids may be referred to as “peptides”.

“Valent” refers to the presence of a specified number of binding sites specific for an antigen in a molecule. As such, the terms “monovalent”, “bivalent”, “tetravalent”, and “hexavalent” refer to the presence of one, two, four and six binding sites, respectively, specific for an antigen in a molecule.

“Subject” includes any human or nonhuman animal. “Nonhuman animal” includes all vertebrates, e.g., mammals and non-mammals, such as nonhuman primates, sheep, dogs, cats, horses, cows, chickens, amphibians, reptiles, etc. Except when noted, the terms “patient” or “subject” are used interchangeably.

“Isolated” refers to a homogenous population of molecules (such as synthetic polynucleotides or a polypeptide such as FN3 domains) which have been substantially separated and/or purified away from other components of the system the molecules are produced in, such as a recombinant cell, as well as a protein that has been subjected to at least one purification or isolation step. “Isolated FN3 domain” refers to an FN3 domain that is substantially free of other cellular material and/or chemicals and encompasses FN3 domains that are isolated to a higher purity, such as to 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% purity.

Compositions of Matter

The present invention provides fibronectin type III (FN3) domains that specifically bind human CD137 protein (SEQ ID NO:44). These molecules can be used in therapeutic and diagnostic applications and in imaging. The present invention provides polynucleotides encoding the FN3 domains of the invention or complementary nucleic acids thereof, vectors, host cells, and methods of making and using them.

The invention provides an isolated FN3 domain that specifically binds CD137.

The FN3 domain of the invention may bind CD137 with a dissociation constant (K_(D)) of less than about 1×10⁻⁷ M, for example less than about 1×10⁻⁸ M, less than about 1×10⁻⁹ M, less than about 1×10⁻¹⁰ M, less than about 1×10⁻¹¹ M, less than about 1×10⁻¹² M, or less than about 1×10⁻¹³ M as determined by surface plasmon resonance or the Kinexa method, as practiced by those of skill in the art. The measured affinity of a particular FN3 domain-antigen interaction can vary if measured under different conditions (e.g., osmolarity, pH). Thus, measurements of affinity and other antigen-binding parameters (e.g., K_(D), K_(on), K_(off)) are made with standardized solutions of protein scaffold and antigen, and a standardized buffer, such as the buffer described herein.

The FN3 domain of the invention may bind CD137 at least 5-fold above the signal obtained for a negative control in standard ELISA assay.

In some embodiments, the FN3 domain that specifically binds CD137 comprises an initiator methionine (Met) linked to the N-terminus of the molecule.

In some embodiments, the FN3 domain that specifically binds CD137 comprises a cysteine (Cys) linked to a C-terminus of the FN3 domain.

The addition of the N-terminal Met and/or the C-terminal Cys may facilitate expression and/or conjugation of half-life extending molecules.

In some embodiments, the FN3 domain that specifically binds CD137 is internalized into a cell.

Internalization of the FN3 domain may facilitate delivery of a cytotoxic agent into tumor cells.

In some embodiments, the FN3 domain that specifically binds CD137 inhibits binding of the CD137 ligand (4-1BBL) to CD137.

Inhibition of binding of 4-1BBL to CD137 by the FN3 domains of the invention may be assessed using competition ELISA. In an exemplary assay, 1 μg/ml recombinant human CD137 is bound on wells of microtiter plates, the wells are washed and blocked, and 10 μg/ml of the test FN3 domain is added. Without washing, 7.5 μg/ml 4-1BBL is added into the wells and incubated for 30 min, after which 0.5 μg/ml anti-4-1BBL antibodies are added and incubated for 30 min. The plates are washed and 0.5 μg/mL neutravidin-HRP conjugate polyclonal antibody is added and incubated for 30 minutes. The plates are washed and POD Chemiluminescence substrate added immediately prior to reading the luminescence signal. The FN3 domains of the invention inhibit binding of 4-1BBL to CD137 when the binding of 4-1BBL is reduced by at least about 80%, 85%, 90%, 95% or 100%.

In some embodiments, the FN3 domain that specifically binds CD137 is a CD137 antagonist.

In some embodiments, the FN3 domain that specifically binds CD137 is a CD137 agonist.

“Antagonist” refers to a FN3 domain that specifically binds CD137 that suppresses at least one activity of CD137 function by inhibiting CD137 binding to to its natural ligand 4-1BB1 or inhibiting CD137 binding to other molecules. A molecule is an antagonist when the at least one reaction or activity is suppressed by at least about 30%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% more than the at least one reaction or activity suppressed in the absence of the antagonist (e.g., negative control), or when the suppression is statistically significant when compared to the suppression in the absence of the antagonist. A typical reaction or activity that is induced by 4-1BBL binding to CD137 is upregulation of survival genes, enhanced cell division, induced cytokine production, and prevention of activation-induced cell death in T cells.

The antagonistic FN3 domains that specifically bind CD137 may be used in the treatment of autoimmune or inflammatory diseases and in general diseases in which suppression of T cell responses is desirable.

“Agonist” refers to a FN3 domain that specifically binds CD137 that induces at least one reaction or activity that is induced by CD137. The FN3 domain is an agonist when the at least one reaction or activity is induced by at least about 30%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% greater than the at least one reaction or activity induced in the absence of the agonist (e.g., negative control), or when the induction is statistically significant when compared to the induction in the absence of the agonist. A typical reaction or activity that is induced by 4-1BBL binding to CD137 is upregulation of survival genes, enhanced cell division, induced cytokine production, and prevention of activation-induced cell death in T cells.

The agonistic FN3 domains that specifically bind CD137 may be used, for example, in the treatment of cancer or viral infections and in general in treatment of diseases in which activation of T cell responses is desirable.

In some embodiments, the FN3 domain that specifically binds CD137 does not inhibit 4-1BBL binding to CD137.

In some embodiments, the FN3 domain that specifically binds CD137 is based on Tencon sequence of SEQ ID NO:1 or Tencon 27 sequence of SEQ ID NO:4, optionally having substitutions at residues positions 11, 14, 17, 37, 46, 73, or 86 (residue numbering corresponding to SEQ ID NO:4).

The invention also provides an isolated FN3 domain that specifically binds CD137 comprising the amino acid sequence of SEQ ID NOs: 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222, 223, or 224.

The invention also provides an isolated FN3 domain that specifically binds CD137 comprising the amino acid sequence of SEQ ID NO:45.

The invention also provides an isolated FN3 domain that specifically binds CD137 comprising the amino acid sequence of SEQ ID NO:46.

The invention also provides an isolated FN3 domain that specifically binds CD137 comprising the amino acid sequence of SEQ ID NO:47.

The invention also provides an isolated FN3 domain that specifically binds CD137 comprising the amino acid sequence of SEQ ID NO:48.

The invention also provides an isolated FN3 domain that specifically binds CD137 comprising the amino acid sequence of SEQ ID NO:49.

The invention also provides an isolated FN3 domain that specifically binds CD137 comprising the amino acid sequence of SEQ ID NO:50.

The invention also provides an isolated FN3 domain that specifically binds CD137 comprising the amino acid sequence of SEQ ID NO:51.

The invention also provides an isolated FN3 domain that specifically binds CD137 comprising the amino acid sequence of SEQ ID NO:52.

The invention also provides an isolated FN3 domain that specifically binds CD137 comprising the amino acid sequence of SEQ ID NO:53.

CD137 comprising the amino acid sequence of SEQ ID NO:54.

The invention also provides an isolated FN3 domain that specifically binds CD137 comprising the amino acid sequence of SEQ ID NO:55.

The invention also provides an isolated FN3 domain that specifically binds CD137 comprising the amino acid sequence of SEQ ID NO:56.

The invention also provides an isolated FN3 domain that specifically binds CD137 comprising the amino acid sequence of SEQ ID NO:57.

The invention also provides an isolated FN3 domain that specifically binds CD137 comprising the amino acid sequence of SEQ ID NO:58.

The invention also provides an isolated FN3 domain that specifically binds CD137 comprising the amino acid sequence of SEQ ID NO:59.

The invention also provides an isolated FN3 domain that specifically binds CD137 comprising the amino acid sequence of SEQ ID NO:60.

The invention also provides an isolated FN3 domain that specifically binds CD137 comprising the amino acid sequence of SEQ ID NO:61.

The invention also provides an isolated FN3 domain that specifically binds CD137 comprising the amino acid sequence of SEQ ID NO:62.

The invention also provides an isolated FN3 domain that specifically binds CD137 comprising the amino acid sequence of SEQ ID NO:63.

The invention also provides an isolated FN3 domain that specifically binds CD137 comprising the amino acid sequence of SEQ ID NO:64.

The invention also provides an isolated FN3 domain that specifically binds CD137 comprising the amino acid sequence of SEQ ID NO:65.

The invention also provides an isolated FN3 domain that specifically binds CD137 comprising the amino acid sequence of SEQ ID NO:66.

The invention also provides an isolated FN3 domain that specifically binds CD137 comprising the amino acid sequence of SEQ ID NO:67.

CD137 comprising the amino acid sequence of SEQ ID NO:68.

The invention also provides an isolated FN3 domain that specifically binds CD137 comprising the amino acid sequence of SEQ ID NO:69.

The invention also provides an isolated FN3 domain that specifically binds CD137 comprising the amino acid sequence of SEQ ID NO:70.

The invention also provides an isolated FN3 domain that specifically binds CD137 comprising the amino acid sequence of SEQ ID NO:71.

The invention also provides an isolated FN3 domain that specifically binds CD137 comprising the amino acid sequence of SEQ ID NO:72.

The invention also provides an isolated FN3 domain that specifically binds CD137 comprising the amino acid sequence of SEQ ID NO:73.

The invention also provides an isolated FN3 domain that specifically binds CD137 comprising the amino acid sequence of SEQ ID NO:74.

The invention also provides an isolated FN3 domain that specifically binds CD137 comprising the amino acid sequence of SEQ ID NO:75.

The invention also provides an isolated FN3 domain that specifically binds CD137 comprising the amino acid sequence of SEQ ID NO:76.

The invention also provides an isolated FN3 domain that specifically binds CD137 comprising the amino acid sequence of SEQ ID NO:77.

The invention also provides an isolated FN3 domain that specifically binds CD137 comprising the amino acid sequence of SEQ ID NO:78.

The invention also provides an isolated FN3 domain that specifically binds CD137 comprising the amino acid sequence of SEQ ID NO:79.

The invention also provides an isolated FN3 domain that specifically binds CD137 comprising the amino acid sequence of SEQ ID NO:80.

The invention also provides an isolated FN3 domain that specifically binds CD137 comprising the amino acid sequence of SEQ ID NO:81.

CD137 comprising the amino acid sequence of SEQ ID NO:82.

The invention also provides an isolated FN3 domain that specifically binds CD137 comprising the amino acid sequence of SEQ ID NO:83.

The invention also provides an isolated FN3 domain that specifically binds CD137 comprising the amino acid sequence of SEQ ID NO:84.

The invention also provides an isolated FN3 domain that specifically binds CD137 comprising the amino acid sequence of SEQ ID NO:85.

The invention also provides an isolated FN3 domain that specifically binds CD137 comprising the amino acid sequence of SEQ ID NO:86.

The invention also provides an isolated FN3 domain that specifically binds CD137 comprising the amino acid sequence of SEQ ID NO:87.

The invention also provides an isolated FN3 domain that specifically binds CD137 comprising the amino acid sequence of SEQ ID NO:88.

The invention also provides an isolated FN3 domain that specifically binds CD137 comprising the amino acid sequence of SEQ ID NO:89.

The invention also provides an isolated FN3 domain that specifically binds CD137 comprising the amino acid sequence of SEQ ID NO:90.

The invention also provides an isolated FN3 domain that specifically binds CD137 comprising the amino acid sequence of SEQ ID NO:91.

The invention also provides an isolated FN3 domain that specifically binds CD137 comprising the amino acid sequence of SEQ ID NO:92.

The invention also provides an isolated FN3 domain that specifically binds CD137 comprising the amino acid sequence of SEQ ID NO:93.

The invention also provides an isolated FN3 domain that specifically binds CD137 comprising the amino acid sequence of SEQ ID NO:94.

The invention also provides an isolated FN3 domain that specifically binds CD137 comprising the amino acid sequence of SEQ ID NO:95.

CD137 comprising the amino acid sequence of SEQ ID NO:96.

The invention also provides an isolated FN3 domain that specifically binds CD137 comprising the amino acid sequence of SEQ ID NO:97.

The invention also provides an isolated FN3 domain that specifically binds CD137 comprising the amino acid sequence of SEQ ID NO:98.

The invention also provides an isolated FN3 domain that specifically binds CD137 comprising the amino acid sequence of SEQ ID NO:99.

The invention also provides an isolated FN3 domain that specifically binds CD137 comprising the amino acid sequence of SEQ ID NO:100.

The invention also provides an isolated FN3 domain that specifically binds CD137 comprising the amino acid sequence of SEQ ID NO:101.

The invention also provides an isolated FN3 domain that specifically binds CD137 comprising the amino acid sequence of SEQ ID NO:102.

The invention also provides an isolated FN3 domain that specifically binds CD137 comprising the amino acid sequence of SEQ ID NO:103.

The invention also provides an isolated FN3 domain that specifically binds CD137 comprising the amino acid sequence of SEQ ID NO:104.

The invention also provides an isolated FN3 domain that specifically binds CD137 comprising the amino acid sequence of SEQ ID NO:105.

The invention also provides an isolated FN3 domain that specifically binds CD137 comprising the amino acid sequence of SEQ ID NO:106.

The invention also provides an isolated FN3 domain that specifically binds CD137 comprising the amino acid sequence of SEQ ID NO:107.

The invention also provides an isolated FN3 domain that specifically binds CD137 comprising the amino acid sequence of SEQ ID NO:108.

The invention also provides an isolated FN3 domain that specifically binds CD137 comprising the amino acid sequence of SEQ ID NO:109.

CD137 comprising the amino acid sequence of SEQ ID NO:110.

The invention also provides an isolated FN3 domain that specifically binds CD137 comprising the amino acid sequence of SEQ ID NO:111.

The invention also provides an isolated FN3 domain that specifically binds CD137 comprising the amino acid sequence of SEQ ID NO:112.

The invention also provides an isolated FN3 domain that specifically binds CD137 comprising the amino acid sequence of SEQ ID NO:113.

The invention also provides an isolated FN3 domain that specifically binds CD137 comprising the amino acid sequence of SEQ ID NO:114.

The invention also provides an isolated FN3 domain that specifically binds CD137 comprising the amino acid sequence of SEQ ID NO:115.

The invention also provides an isolated FN3 domain that specifically binds CD137 comprising the amino acid sequence of SEQ ID NO:116.

The invention also provides an isolated FN3 domain that specifically binds CD137 comprising the amino acid sequence of SEQ ID NO:117.

The invention also provides an isolated FN3 domain that specifically binds CD137 comprising the amino acid sequence of SEQ ID NO:118.

The invention also provides an isolated FN3 domain that specifically binds CD137 comprising the amino acid sequence of SEQ ID NO:119.

The invention also provides an isolated FN3 domain that specifically binds CD137 comprising the amino acid sequence of SEQ ID NO:120.

The invention also provides an isolated FN3 domain that specifically binds CD137 comprising the amino acid sequence of SEQ ID NO:121.

The invention also provides an isolated FN3 domain that specifically binds CD137 comprising the amino acid sequence of SEQ ID NO:122.

The invention also provides an isolated FN3 domain that specifically binds CD137 comprising the amino acid sequence of SEQ ID NO:123.

CD137 comprising the amino acid sequence of SEQ ID NO:124.

The invention also provides an isolated FN3 domain that specifically binds CD137 comprising the amino acid sequence of SEQ ID NO:125.

The invention also provides an isolated FN3 domain that specifically binds CD137 comprising the amino acid sequence of SEQ ID NO:126.

The invention also provides an isolated FN3 domain that specifically binds CD137 comprising the amino acid sequence of SEQ ID NO:127.

The invention also provides an isolated FN3 domain that specifically binds CD137 comprising the amino acid sequence of SEQ ID NO:128.

The invention also provides an isolated FN3 domain that specifically binds CD137 comprising the amino acid sequence of SEQ ID NO:129.

The invention also provides an isolated FN3 domain that specifically binds CD137 comprising the amino acid sequence of SEQ ID NO:130.

The invention also provides an isolated FN3 domain that specifically binds CD137 comprising the amino acid sequence of SEQ ID NO:131.

The invention also provides an isolated FN3 domain that specifically binds CD137 comprising the amino acid sequence of SEQ ID NO:132.

The invention also provides an isolated FN3 domain that specifically binds CD137 comprising the amino acid sequence of SEQ ID NO:133.

The invention also provides an isolated FN3 domain that specifically binds CD137 comprising the amino acid sequence of SEQ ID NO:134.

The invention also provides an isolated FN3 domain that specifically binds CD137 comprising the amino acid sequence of SEQ ID NO:135.

The invention also provides an isolated FN3 domain that specifically binds CD137 comprising the amino acid sequence of SEQ ID NO:136.

The invention also provides an isolated FN3 domain that specifically binds CD137 comprising the amino acid sequence of SEQ ID NO:137.

CD137 comprising the amino acid sequence of SEQ ID NO:138.

The invention also provides an isolated FN3 domain that specifically binds CD137 comprising the amino acid sequence of SEQ ID NO:139.

The invention also provides an isolated FN3 domain that specifically binds CD137 comprising the amino acid sequence of SEQ ID NO:140.

The invention also provides an isolated FN3 domain that specifically binds CD137 comprising the amino acid sequence of SEQ ID NO:141.

The invention also provides an isolated FN3 domain that specifically binds CD137 comprising the amino acid sequence of SEQ ID NO:142.

The invention also provides an isolated FN3 domain that specifically binds CD137 comprising the amino acid sequence of SEQ ID NO:143.

The invention also provides an isolated FN3 domain that specifically binds CD137 comprising the amino acid sequence of SEQ ID NO:144.

The invention also provides an isolated FN3 domain that specifically binds CD137 comprising the amino acid sequence of SEQ ID NO:145.

The invention also provides an isolated FN3 domain that specifically binds CD137 comprising the amino acid sequence of SEQ ID NO:146.

The invention also provides an isolated FN3 domain that specifically binds CD137 comprising the amino acid sequence of SEQ ID NO:147.

The invention also provides an isolated FN3 domain that specifically binds CD137 comprising the amino acid sequence of SEQ ID NO:148.

The invention also provides an isolated FN3 domain that specifically binds CD137 comprising the amino acid sequence of SEQ ID NO:149.

The invention also provides an isolated FN3 domain that specifically binds CD137 comprising the amino acid sequence of SEQ ID NO:150.

The invention also provides an isolated FN3 domain that specifically binds CD137 comprising the amino acid sequence of SEQ ID NO:151.

CD137 comprising the amino acid sequence of SEQ ID NO:152.

The invention also provides an isolated FN3 domain that specifically binds CD137 comprising the amino acid sequence of SEQ ID NO:153.

The invention also provides an isolated FN3 domain that specifically binds CD137 comprising the amino acid sequence of SEQ ID NO:154.

The invention also provides an isolated FN3 domain that specifically binds CD137 comprising the amino acid sequence of SEQ ID NO:155.

The invention also provides an isolated FN3 domain that specifically binds CD137 comprising the amino acid sequence of SEQ ID NO:156.

The invention also provides an isolated FN3 domain that specifically binds CD137 comprising the amino acid sequence of SEQ ID NO:157.

The invention also provides an isolated FN3 domain that specifically binds CD137 comprising the amino acid sequence of SEQ ID NO:158.

The invention also provides an isolated FN3 domain that specifically binds CD137 comprising the amino acid sequence of SEQ ID NO:159.

The invention also provides an isolated FN3 domain that specifically binds CD137 comprising the amino acid sequence of SEQ ID NO:160.

The invention also provides an isolated FN3 domain that specifically binds CD137 comprising the amino acid sequence of SEQ ID NO:161.

The invention also provides an isolated FN3 domain that specifically binds CD137 comprising the amino acid sequence of SEQ ID NO:162.

The invention also provides an isolated FN3 domain that specifically binds CD137 comprising the amino acid sequence of SEQ ID NO:163.

The invention also provides an isolated FN3 domain that specifically binds CD137 comprising the amino acid sequence of SEQ ID NO:164.

The invention also provides an isolated FN3 domain that specifically binds CD137 comprising the amino acid sequence of SEQ ID NO:165.

CD137 comprising the amino acid sequence of SEQ ID NO:166.

The invention also provides an isolated FN3 domain that specifically binds CD137 comprising the amino acid sequence of SEQ ID NO:167.

The invention also provides an isolated FN3 domain that specifically binds CD137 comprising the amino acid sequence of SEQ ID NO:168.

The invention also provides an isolated FN3 domain that specifically binds CD137 comprising the amino acid sequence of SEQ ID NO:169.

The invention also provides an isolated FN3 domain that specifically binds CD137 comprising the amino acid sequence of SEQ ID NO:170.

The invention also provides an isolated FN3 domain that specifically binds CD137 comprising the amino acid sequence of SEQ ID NO:171.

The invention also provides an isolated FN3 domain that specifically binds CD137 comprising the amino acid sequence of SEQ ID NO:172.

The invention also provides an isolated FN3 domain that specifically binds CD137 comprising the amino acid sequence of SEQ ID NO:173.

The invention also provides an isolated FN3 domain that specifically binds CD137 comprising the amino acid sequence of SEQ ID NO:174.

The invention also provides an isolated FN3 domain that specifically binds CD137 comprising the amino acid sequence of SEQ ID NO:175.

The invention also provides an isolated FN3 domain that specifically binds CD137 comprising the amino acid sequence of SEQ ID NO:176.

The invention also provides an isolated FN3 domain that specifically binds CD137 comprising the amino acid sequence of SEQ ID NO:177.

The invention also provides an isolated FN3 domain that specifically binds CD137 comprising the amino acid sequence of SEQ ID NO:178.

The invention also provides an isolated FN3 domain that specifically binds CD137 comprising the amino acid sequence of SEQ ID NO:179.

CD137 comprising the amino acid sequence of SEQ ID NO:180.

The invention also provides an isolated FN3 domain that specifically binds CD137 comprising the amino acid sequence of SEQ ID NO:181.

The invention also provides an isolated FN3 domain that specifically binds CD137 comprising the amino acid sequence of SEQ ID NO:182.

The invention also provides an isolated FN3 domain that specifically binds CD137 comprising the amino acid sequence of SEQ ID NO:183.

The invention also provides an isolated FN3 domain that specifically binds CD137 comprising the amino acid sequence of SEQ ID NO:184.

The invention also provides an isolated FN3 domain that specifically binds CD137 comprising the amino acid sequence of SEQ ID NO:185.

The invention also provides an isolated FN3 domain that specifically binds CD137 comprising the amino acid sequence of SEQ ID NO:186.

The invention also provides an isolated FN3 domain that specifically binds CD137 comprising the amino acid sequence of SEQ ID NO:187.

The invention also provides an isolated FN3 domain that specifically binds CD137 comprising the amino acid sequence of SEQ ID NO:188.

The invention also provides an isolated FN3 domain that specifically binds CD137 comprising the amino acid sequence of SEQ ID NO:189.

The invention also provides an isolated FN3 domain that specifically binds CD137 comprising the amino acid sequence of SEQ ID NO:190.

The invention also provides an isolated FN3 domain that specifically binds CD137 comprising the amino acid sequence of SEQ ID NO:191.

The invention also provides an isolated FN3 domain that specifically binds CD137 comprising the amino acid sequence of SEQ ID NO:192.

The invention also provides an isolated FN3 domain that specifically binds CD137 comprising the amino acid sequence of SEQ ID NO:193.

CD137 comprising the amino acid sequence of SEQ ID NO:194.

The invention also provides an isolated FN3 domain that specifically binds CD137 comprising the amino acid sequence of SEQ ID NO:195.

The invention also provides an isolated FN3 domain that specifically binds CD137 comprising the amino acid sequence of SEQ ID NO:196.

The invention also provides an isolated FN3 domain that specifically binds CD137 comprising the amino acid sequence of SEQ ID NO:197.

The invention also provides an isolated FN3 domain that specifically binds CD137 comprising the amino acid sequence of SEQ ID NO:198.

The invention also provides an isolated FN3 domain that specifically binds CD137 comprising the amino acid sequence of SEQ ID NO:199.

The invention also provides an isolated FN3 domain that specifically binds CD137 comprising the amino acid sequence of SEQ ID NO:200.

The invention also provides an isolated FN3 domain that specifically binds CD137 comprising the amino acid sequence of SEQ ID NO:201.

The invention also provides an isolated FN3 domain that specifically binds CD137 comprising the amino acid sequence of SEQ ID NO:202.

The invention also provides an isolated FN3 domain that specifically binds CD137 comprising the amino acid sequence of SEQ ID NO:203.

The invention also provides an isolated FN3 domain that specifically binds CD137 comprising the amino acid sequence of SEQ ID NO:204.

The invention also provides an isolated FN3 domain that specifically binds CD137 comprising the amino acid sequence of SEQ ID NO:205.

The invention also provides an isolated FN3 domain that specifically binds CD137 comprising the amino acid sequence of SEQ ID NO:206.

The invention also provides an isolated FN3 domain that specifically binds CD137 comprising the amino acid sequence of SEQ ID NO:207.

CD137 comprising the amino acid sequence of SEQ ID NO:208.

The invention also provides an isolated FN3 domain that specifically binds CD137 comprising the amino acid sequence of SEQ ID NO:209.

The invention also provides an isolated FN3 domain that specifically binds CD137 comprising the amino acid sequence of SEQ ID NO:210.

The invention also provides an isolated FN3 domain that specifically binds CD137 comprising the amino acid sequence of SEQ ID NO:211.

The invention also provides an isolated FN3 domain that specifically binds CD137 comprising the amino acid sequence of SEQ ID NO:212.

The invention also provides an isolated FN3 domain that specifically binds CD137 comprising the amino acid sequence of SEQ ID NO:213.

The invention also provides an isolated FN3 domain that specifically binds CD137 comprising the amino acid sequence of SEQ ID NO:214.

The invention also provides an isolated FN3 domain that specifically binds CD137 comprising the amino acid sequence of SEQ ID NO:215.

The invention also provides an isolated FN3 domain that specifically binds CD137 comprising the amino acid sequence of SEQ ID NO:216.

The invention also provides an isolated FN3 domain that specifically binds CD137 comprising the amino acid sequence of SEQ ID NO:217.

The invention also provides an isolated FN3 domain that specifically binds CD137 comprising the amino acid sequence of SEQ ID NO:218.

The invention also provides an isolated FN3 domain that specifically binds CD137 comprising the amino acid sequence of SEQ ID NO:219.

The invention also provides an isolated FN3 domain that specifically binds CD137 comprising the amino acid sequence of SEQ ID NO:220.

The invention also provides an isolated FN3 domain that specifically binds CD137 comprising the amino acid sequence of SEQ ID NO:221.

CD137 comprising the amino acid sequence of SEQ ID NO:222.

The invention also provides an isolated FN3 domain that specifically binds CD137 comprising the amino acid sequence of SEQ ID NO:223.

The invention also provides an isolated FN3 domain that specifically binds CD137 comprising the amino acid sequence of SEQ ID NO:224.

In some embodiments, the isolated FN3 domain that specifically binds CD137 comprises an initiator methionine (Met) linked to the N-terminus of the molecule.

In some embodiments, the isolated FN3 domain that specifically binds CD137 comprises an amino acid sequence that is 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to one of the amino acid sequences of SEQ ID NOs: 45-224.

Conjugates of the FN3 Domains that Specifically Bind CD137 of the Invention

The invention also provides an isolated FN3 domain that specifically binds CD137 conjugated to a heterologous molecule(s).

In some embodiments, the heterologous molecule is a detectable label or a cytotoxic agent.

The invention also provides an FN3 domain that specifically binds CD137 conjugated to a detectable label.

The invention also provides an FN3 domain that specifically binds CD137 conjugated to a cytotoxic agent.

In some embodiments, the detectable label is also a cytotoxic agent.

The FN3 domains that specifically bind CD137 of the invention conjugated to a detectable label can be used to evaluate expression of CD137 on samples such as tumor tissue in vivo or in vitro.

Detectable label includes compositions that when conjugated to the FN3 domains that specifically bind CD137 of the invention renders the latter detectable, via spectroscopic, photochemical, biochemical, immunochemical, or chemical means.

Exemplary detectable labels include radioactive isotopes, magnetic beads, metallic beads, colloidal particles, fluorescent dyes, electron-dense reagents, enzymes (for example, as commonly used in an ELISA), biotin, digoxigenin, haptens, luminescent molecules, chemiluminescent molecules, fluorochromes, fluorophores, fluorescent quenching agents, colored molecules, radioactive isotopes, cintillants, avidin, streptavidin, protein A, protein G, antibodies or fragments thereof, polyhistidine, Ni²⁺, Flag tags, myc tags, heavy metals, enzymes, alkaline phosphatase, peroxidase, luciferase, electron donors/acceptors, acridinium esters, and colorimetric substrates.

A detectable label may emit a signal spontaneously, such as when the detectable label is a radioactive isotope. In other cases the detectable label emits a signal as a result of being stimulated by an external field.

Exemplary radioactive isotopes may be γ-emitting, Auger-emitting, β-emitting, an alpha-emitting or positron-emitting radioactive isotope. Exemplary radioactive isotopes include ³H, ¹¹C, ¹³C, ¹⁵N, ¹⁸F, ¹⁹F, ⁵⁵Co, ⁵⁷Co, ⁶⁰Co, ⁶¹Cu, ⁶²Cu, ⁶⁴Cu, ⁶⁷Cu, ⁶⁸Ga, ⁷²As, ⁷⁵Br, ⁸⁶Y, ⁸⁹Zr, ⁹⁰Sr, ^(94m)Tc, ^(99m)Tc, ¹¹⁵In, ¹²³I, ¹²⁴I, ¹²⁵I, ¹³¹I, ²¹¹At, ²¹²Bi, ²¹³Bi, ²²³Ra, ²²⁶Ra, ²²⁵Ac and ²²⁷Ac.

Exemplary metal atoms are metals with an atomic number greater than 20, such as calcium atoms, scandium atoms, titanium atoms, vanadium atoms, chromium atoms, manganese atoms, iron atoms, cobalt atoms, nickel atoms, copper atoms, zinc atoms, gallium atoms, germanium atoms, arsenic atoms, selenium atoms, bromine atoms, krypton atoms, rubidium atoms, strontium atoms, yttrium atoms, zirconium atoms, niobium atoms, molybdenum atoms, technetium atoms, ruthenium atoms, rhodium atoms, palladium atoms, silver atoms, cadmium atoms, indium atoms, tin atoms, antimony atoms, tellurium atoms, iodine atoms, xenon atoms, cesium atoms, barium atoms, lanthanum atoms, hafnium atoms, tantalum atoms, tungsten atoms, rhenium atoms, osmium atoms, iridium atoms, platinum atoms, gold atoms, mercury atoms, thallium atoms, lead atoms, bismuth atoms, francium atoms, radium atoms, actinium atoms, cerium atoms, praseodymium atoms, neodymium atoms, promethium atoms, samarium atoms, europium atoms, gadolinium atoms, terbium atoms, dysprosium atoms, holmium atoms, erbium atoms, thulium atoms, ytterbium atoms, lutetium atoms, thorium atoms, protactinium atoms, uranium atoms, neptunium atoms, plutonium atoms, americium atoms, curium atoms, berkelium atoms, californium atoms, einsteinium atoms, fermium atoms, mendelevium atoms, nobelium atoms, or lawrencium atoms.

In some embodiments, the metal atoms may be alkaline earth metals with an atomic number greater than twenty.

In some embodiments, the metal atoms may be lanthanides.

In some embodiments, the metal atoms may be actinides.

In some embodiments, the metal atoms may be transition metals.

In some embodiments, the metal atoms may be poor metals.

In some embodiments, the metal atoms may be gold atoms, bismuth atoms, tantalum atoms, and gadolinium atoms.

In some embodiments, the metal atoms may be metals with an atomic number of 53 (i.e., iodine) to 83 (i.e., bismuth).

In some embodiments, the metal atoms may be atoms suitable for magnetic resonance imaging.

The metal atoms may be metal ions in the form of +1, +2, or +3 oxidation states, such as Ba²⁺, Bi³⁺, Cs⁺, Ca²⁺, Cr²⁺, Cr³⁺, Cr⁶⁺, Co²⁺, Co³⁺, Cu⁺, Cu²⁺, Cu³⁺, Ga³⁺, Gd³⁺, Au⁺, Au³⁺, Fe²⁺, Fe³⁺, F³⁺, Pb²⁺, Mn²⁺, Mn³⁺, Mn⁴⁺, Mn⁷⁺, Hg²⁺, Ni²⁺, Ni³⁺, Ag⁺, Sr²⁺, Sn²⁺, Sn⁴⁺, and Zn²⁺. The metal atoms may comprise a metal oxide, such as iron oxide, manganese oxide, or gadolinium oxide.

Suitable dyes include any commercially available dyes such as, for example, 5(6)-carboxyfluorescein, IRDye 680RD maleimide or IRDye 800CW, ruthenium polypyridyl dyes, and the like.

Suitable fluorophores are fluorescein isothiocyante (FITC), fluorescein thiosemicarbazide, rhodamine, Texas Red, CyDyes (e.g., Cy3, Cy5, Cy5.5), Alexa Fluors (e.g., Alexa488, Alexa555, Alexa594; Alexa647), near infrared (NIR) (700-900 nm) fluorescent dyes, and carbocyanine and aminostyryl dyes.

The FN3 domains that specifically bind CD137 conjugated to a detectable label may be used as an imaging agent to evaluate tumor distribution, diagnosis for the presence of tumor cells and/or, recurrence of tumor.

In some embodiments, the FN3 domains that specifically bind CD137 of the invention are conjugated to a cytotoxic agent.

In some embodiments, the cytotoxic agent is a chemotherapeutic agent, a drug, a growth inhibitory agent, a toxin (e.g., an enzymatically active toxin of bacterial, fungal, plant, or animal origin, or fragments thereof), or a radioactive isotope (i.e., a radioconjugate).

The FN3 domains that specifically bind CD137 conjugated to a cytotoxic agent of the invention may be used in the targeted delivery of the cytotoxic agent to CD137 expressing tumor cell, and intracellular accumulation therein, wherein systemic administration of these unconjugated cytotoxic agents may result in unacceptable levels of toxicity to normal cells.

In some embodiments, the cytotoxic agent is daunomycin, doxorubicin, methotrexate, vindesine, bacterial toxins such as diphtheria toxin, ricin, geldanamycin, maytansinoids or calicheamicin. The cytotoxic agent may elict their cytotoxic and cytostatic effects by mechanisms including tubulin binding, DNA binding, or topoisomerase inhibition.

In some embodiments, the cytotoxic agent is an enzymatically active toxins such as diphtheria A chain, nonbinding active fragments of diphtheria toxin, exotoxin A chain (from Pseudomonas aeruginosa), ricin A chain, abrin A chain, modeccin A chain, alpha-sarcin, Aleurites fordii proteins, dianthin proteins, Phytolaca americana proteins (PAPI, PAPII, and PAP-S), Momordica charantia inhibitor, curcin, crotin, Sapaonaria officinalis inhibitor, gelonin, mitogellin, restrictocin, phenomycin, enomycin, and the tricothecenes.

In some embodiments, the cytotoxic agent is a radionuclide, such as ²¹²Bi, ¹³¹I, ¹³¹In, ⁹⁰Y, and ¹⁸⁶Re.

In some embodiments, the cytotoxic agent is dolastatins or dolostatin peptidic analogs and derivatives, auristatin or monomethyl auristatin phenylalanine. Exemplary molecules are disclosed in U.S. Pat. Nos. 5,635,483 and 5,780,588. Dolastatins and auristatins have been shown to interfere with microtubule dynamics, GTP hydrolysis, and nuclear and cellular division (Woyke et al (2001) Antimicrob Agents and Chemother. 45(12):3580-3584) and have anticancerand antifungal activity. The dolastatin or auristatin drug moiety may be attached to the FN3 domain of the invention through the N (amino) terminus or the C (carboxyl) terminus of the peptidic drug moiety (WO 02/088172), or via any cysteine engineered into the FN3 domain.

The FN3 domains that specifically bind CD137 of the invention may be conjugated to a detectable label using known methods.

In some embodiments, the detectable label is complexed with a chelating agent.

In some embodiments, the detectable label is conjugated to the FN3 domain that specifically binds CD137 of the invention via a linker.

The detectable label or the cytotoxic moiety may be linked directly, or indirectly, to the FN3 domain that specifically binds CD137 of the invention using known methods. Suitable linkers are known in the art and include, for example, prosthetic groups, non-phenolic linkers (derivatives of N-succimidyl-benzoates; dodecaborate), chelating moieties of both macrocyclics and acyclic chelators, such as derivatives of 1,4,7,10-tetraazacyclododecane-1,4,7,10,tetraacetic acid (DOTA), derivatives of diethylenetriaminepentaacetic avid (DTPA), derivatives of S-2-(4-Isothiocyanatobenzyl)-1,4,7-triazacyclononane-1,4,7-triacetic acid (NOTA) and derivatives of 1,4,8,11-tetraazacyclodocedan-1,4,8,11-tetraacetic acid (TETA), N-succinimidyl-3-(2-pyridyldithiol) propionate (SPDP), iminothiolane (IT), bifunctional derivatives of imidoesters (such as dimethyl adipimidate HCl), active esters (such as disuccinimidyl suberate), aldehydes (such as glutaraldehyde), bis-azido compounds (such as bis(p-azidobenzoyl)hexanediamine), bis-diazonium derivatives (such as bis-(p-diazoniumbenzoyl)-ethylenediamine), diisocyanates (such as toluene 2,6-diisocyanate), and bis-active fluorine compounds (such as 1,5-difluoro-2,4-dinitrobenzene) and other chelating moieties. Suitable peptide linkers are well known.

In some embodiment, the FN3 domain that specifically binds CD137 is removed from the blood via renal clearance.

Isolation of CD137 Binding FN3 Domains from a Library Based on Tencon Sequence

Tencon (SEQ ID NO:1) is a non-naturally occurring fibronectin type III (FN3) domain designed from a consensus sequence of fifteen FN3 domains from human tenascin-C (Jacobs et al., Protein Engineering, Design, and Selection, 25:107-117, 2012; U.S. Pat. Publ. No. 2010/0216708). The crystal structure of Tencon shows six surface-exposed loops that connect seven beta-strands as is characteristic to the FN3 domains, the beta-strands referred to as A, B, C, D, E, F, and G, and the loops referred to as AB, BC, CD, DE, EF, and FG loops (Bork and Doolittle, Proc Natl Acad Sci USA 89:8990-8992, 1992; U.S. Pat. No. 6,673,901). These loops, or selected residues within each loop, may be randomized in order to construct libraries of fibronectin type III (FN3) domains that may be used to select novel molecules that bind CD137. Table 1 shows positions and sequences of each loop and beta-strand in Tencon (SEQ ID NO:1).

Library designed based on Tencon sequence may thus have randomized FG loop, or randomized BC and FG loops, such as libraries TCL1 or TCL2 as described below. The Tencon BC loop is 7 amino acids long, thus 1, 2, 3, 4, 5, 6 or 7 amino acids may be randomized in the library diversified at the BC loop and designed based on Tencon sequence. The Tencon FG loop is 7 amino acids long, thus 1, 2, 3, 4, 5, 6 or 7 amino acids may be randomized in the library diversified at the FG loop and designed based on Tencon sequence. Further diversity at loops in the Tencon libraries may be achieved by insertion and/or deletions of residues at loops. For example, the FG and/or BC loops may be extended by 1-22 amino acids, or decreased by 1-3 amino acids. The FG loop in Tencon is 7 amino acids long, whereas the corresponding loop in antibody heavy chains ranges from 4-28 residues. To provide maximum diversity, the FG loop may be diversified in sequence as well as in length to correspond to the antibody CDR3 length range of 4-28 residues. For example, the FG loop can further be diversified in length by extending the loop by additional 1, 2, 3, 4 or 5 amino acids.

Library designed based on Tencon sequence may also have randomized alternative surfaces that form on a side of the FN3 domain and comprise two or more beta strands, and at least one loop. One such alternative surface is formed by amino acids in the C and the F beta-strands and the CD and the FG loops (a C-CD-F-FG surface). A library design based on Tencon alternative C-CD-F-FG surface is described in U.S. Pat. Publ. No. 2013/0226834. Library designed based on Tencon sequence also includes libraries designed based on Tencon variants, such as Tencon variants having substitutions at residues positions 11, 14, 17, 37, 46, 73, or 86 (residue numbering corresponding to SEQ ID NO:1), and which variants display improve thermal stability. Exemplary Tencon variants are described in US Pat. Publ. No. 2011/0274623, and include Tencon27 (SEQ ID NO:4) having substitutions E11R, L17A, N46V and E86I when compared to Tencon of SEQ ID NO:1.

TABLE 1 Tencon topology Tencon FN3 domain (SEQ ID NO: 1) A strand  1-12 AB loop 13-16 B strand 17-21 BC loop 22-28 C strand 29-37 CD loop 38-43 D strand 44-50 DE loop 51-54 E strand 55-59 EF loop 60-64 F strand 65-74 FG loop 75-81 G strand 82-89

Tencon and other FN3 sequence based libraries may be randomized at chosen residue positions using a random or defined set of amino acids. For example, variants in the library having random substitutions may be generated using NNK codons, which encode all 20 naturally occurring amino acids. In other diversification schemes, DVK codons may be used to encode amino acids Ala, Trp, Tyr, Lys, Thr, Asn, Lys, Ser, Arg, Asp, Glu, Gly, and Cys. Alternatively, NNS codons may be used to give rise to all 20 amino acid residues and simultaneously reducing the frequency of stop codons. Libraries of FN3 domains with biased amino acid distribution at positions to be diversified may be synthesized for example using Slonomics® technology (http:_//www_sloning_com). This technology uses a library of pre-made double stranded triplets that act as universal building blocks sufficient for thousands of gene synthesis processes. The triplet library represents all possible sequence combinations necessary to build any desired DNA molecule. The codon designations are according to the well-known IUB code.

The FN3 domains that specifically bind CD137 of the invention may be isolated by producing the FN3 library such as the Tencon library using cis display to ligate DNA fragments encoding the scaffold proteins to a DNA fragment encoding RepA to generate a pool of protein-DNA complexes formed after in vitro translation wherein each protein is stably associated with the DNA that encodes it (U.S. Pat. No. 7,842,476; Odegrip et al., Proc Natl Acad Sci USA 101, 2806-2810, 2004), and assaying the library for specific binding to PSMA by any method known in the art and described in the Example. Exemplary well known methods which can be used are ELISA, sandwich immunoassays, and competitive and non-competitive assays (see, e.g., Ausubel et al., eds, 1994, Current Protocols in Molecular Biology, Vol. 1, John Wiley & Sons, Inc., New York). The identified FN3 domains that specifically bind CD137 are further characterized for their binding to CD137, modulation of CD137 activity, internalization, stability, and other desired characteristics.

The FN3 domains that specifically bind CD137 of the invention may be generated using any FN3 domain as a template to generate a library and screening the library for molecules specifically binding CD137 using methods provided within. Exemplar FN3 domains that may be used are the 3rd FN3 domain of tenascin C (TN3) (SEQ ID NO:32), Fibcon (SEQ ID NO:33), and the 10^(th) FN3 domain of fibronectin (FN10) (SEQ ID NO:34). Standard cloning and expression techniques are used to clone the libraries into a vector or synthesize double stranded cDNA cassettes of the library, to express, or to translate the libraries in vitro. For example ribosome display (Hanes and Pluckthun, Proc Natl Acad Sci USA, 94, 4937-4942, 1997), mRNA display (Roberts and Szostak, Proc Natl Acad Sci USA, 94, 12297-12302, 1997), or other cell-free systems (U.S. Pat. No. 5,643,768) can be used. The libraries of the FN3 domain variants may be expressed as fusion proteins displayed on the surface for example of any suitable bacteriophage. Methods for displaying fusion polypeptides on the surface of a bacteriophage are well known (U.S. Pat. Publ. No. 2011/0118144; Int. Pat. Publ. No. WO2009/085462; U.S. Pat. Nos. 6,969,108; 6,172,197; 5,223,409; 6,582,915; 6,472,147).

In some embodiments. the FN3 domain that specifically binds CD137 is based on Tencon sequence of SEQ ID NO:1 or Tencon27 sequence of SEQ ID NO:4, the SEQ ID NO:1 or the SEQ ID NO:4, optionally having substitutions at residues positions 11, 14, 17, 37, 46, 73, and/or 86.

The FN3 domains that specifically bind CD137 of the invention may be modified to improve their properties such as improve thermal stability and reversibility of thermal folding and unfolding. Several methods have been applied to increase the apparent thermal stability of proteins and enzymes, including rational design based on comparison to highly similar thermostable sequences, design of stabilizing disulfide bridges, mutations to increase alpha-helix propensity, engineering of salt bridges, alteration of the surface charge of the protein, directed evolution, and composition of consensus sequences (Lehmann and Wyss, Curr Opin Biotechnol, 12, 371-375, 2001). High thermal stability may increase the yield of the expressed protein, improve solubility or activity, decrease immunogenicity, and minimize the need of a cold chain in manufacturing. Residues that may be substituted to improve thermal stability of Tencon (SEQ ID NO:1) are residue positions 11, 14, 17, 37, 46, 73, or 86, and are described in US Pat. Publ. No. 2011/0274623. Substitutions corresponding to these residues may be incorporated to the FN3 domain containing molecules of the invention.

Measurement of protein stability and protein lability can be viewed as the same or different aspects of protein integrity. Proteins are sensitive or “labile” to denaturation caused by heat, by ultraviolet or ionizing radiation, changes in the ambient osmolarity and pH if in liquid solution, mechanical shear force imposed by small pore-size filtration, ultraviolet radiation, ionizing radiation, such as by gamma irradiation, chemical or heat dehydration, or any other action or force that may cause protein structure disruption. The stability of the molecule can be determined using standard methods. For example, the stability of a molecule can be determined by measuring the thermal melting (“T_(m)”) temperature, the temperature in ° Celsius (° C.) at which half of the molecules become unfolded, using standard methods. Typically, the higher the T_(m), the more stable the molecule. In addition to heat, the chemical environment also changes the ability of the protein to maintain a particular three dimensional structure.

In one embodiment, the FN3 domain that specifically binds CD137 of the invention may exhibit increased stability by at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% or more compared to the same domain prior to engineering measured by the increase in the T_(m).

Chemical denaturation can likewise be measured by a variety of methods. Chemical denaturants include guanidinium hydrochloride, guanidinium thiocyanate, urea, acetone, organic solvents (DMF, benzene, acetonitrile), salts (ammonium sulfate, lithium bromide, lithium chloride, sodium bromide, calcium chloride, sodium chloride); reducing agents (e.g. dithiothreitol, beta-mercaptoethanol, dinitrothiobenzene, and hydrides, such as sodium borohydride), non-ionic and ionic detergents, acids (e.g. hydrochloric acid (HCl), acetic acid (CH₃COOH), halogenated acetic acids), hydrophobic molecules (e.g. phosopholipids), and targeted denaturants. Quantitation of the extent of denaturation can rely on loss of a functional property, such as ability to bind a target molecule, or by physiochemical properties, such as tendency to aggregation, exposure of formerly solvent inaccessible residues, or disruption or formation of disulfide bonds.

The FN3 domain that specifically binds CD137 may be generated as monomers, dimers, or multimers, for example, as a means to increase the valency and thus the avidity of target molecule binding, or to generate bi- or multispecific scaffolds simultaneously binding two or more different target molecules. The dimers and multimers may be generated by linking monospecific, bi- or multispecific protein scaffolds, for example, by the inclusion of an amino acid linker, for example a linker containing poly-glycine, glycine and serine, or alanine and proline. Exemplary linker include (GS)₂, (SEQ ID NO:35), (GGGS)₂ (SEQ ID NO:36), (GGGGS)₅ (SEQ ID NO:37), (AP)₂ (SEQ ID NO:38), (AP)₅ (SEQ ID NO:39), (AP)₁₀ (SEQ ID NO:40), (AP)₂₀ (SEQ ID NO:41) and A(EAAAK)₅AAA (SEQ ID NO:42). The dimers and multimers may be linked to each other in a N- to C-direction. The use of naturally occurring as well as artificial peptide linkers to connect polypeptides into novel linked fusion polypeptides is well known in the literature (Hallewell et al., J Biol Chem 264, 5260-5268, 1989; Alfthan et al., Protein Eng. 8, 725-731, 1995; Robinson & Sauer, Biochemistry 35, 109-116, 1996; U.S. Pat. No. 5,856,456).

Half-Life Extending Moieties

The FN3 domains that specifically bind CD137 may incorporate other subunits for example via covalent interaction. In one aspect of the invention, the FN3 domains that specifically bind CD137 further comprise a half-life extending moiety. Exemplary half-life extending moieties are albumin, albumin variants, albumin-binding proteins and/or domains, transferrin and fragments and analogues thereof, and Fc regions. An exemplary albumin variant is shown in SEQ ID NO:43. Amino acid sequences of the human Fc regions are well known, and include IgG1, IgG2, IgG3, IgG4, IgM, IgA and IgE Fc regions.

All or a portion of an antibody constant region may be attached to the FN3 domain that specifically binds CD137 to impart antibody-like properties, especially those properties associated with the Fc region, such as Fc effector functions such as Clq binding, complement dependent cytotoxicity (CDC), Fc receptor binding, antibody-dependent cell-mediated cytotoxicity (ADCC), phagocytosis, down regulation of cell surface receptors (e.g., B cell receptor; BCR), and may be further modified by modifying residues in the Fc responsible for these activities (for review; see Strohl, Curr Opin Biotechnol. 20, 685-691, 2009).

Additional moieties may be incorporated into the FN3 domains that specifically bind CD137 such as polyethylene glycol (PEG) molecules, such as PEG5000 or PEG20,000, fatty acids and fatty acid esters of different chain lengths, for example laurate, myristate, stearate, arachidate, behenate, oleate, arachidonate, octanedioic acid, tetradecanedioic acid, octadecanedioic acid, docosanedioic acid, and the like, polylysine, octane, carbohydrates (dextran, cellulose, oligo- or polysaccharides) for desired properties. These moieties may be direct fusions with the protein scaffold coding sequences and may be generated by standard cloning and expression techniques. Alternatively, well known chemical coupling methods may be used to attach the moieties to recombinantly produced molecules of the invention.

A pegyl moiety may for example be added to the FN3 domain that specifically binds CD137 by incorporating a cysteine residue to the C-terminus of the molecule, or engineering cysteines into residue positions that face away from the CD137 binding face of the molecule, and attaching a pegyl group to the cysteine using well known methods.

FN3 domains that specifically bind CD137 incorporating additional moieties may be compared for functionality by several well-known assays. For example, altered properties due to incorporation of Fc domains and/or Fc domain variants may be assayed in Fc receptor binding assays using soluble forms of the receptors, such as the FcγRT, FcγRII, FcγRIII or FcRn receptors, or using well known cell-based assays measuring for example ADCC or CDC, or evaluating pharmacokinetic properties of the molecules of the invention in in vivo models.

Polynucleotides, Vectors, Host Cells

The invention also provides nucleic acids encoding the FN3 domains specifically binding CD137 as isolated polynucleotides or as portions of expression vectors or as portions of linear DNA sequences, including linear DNA sequences used for in vitro transcription/translation, vectors compatible with prokaryotic, eukaryotic or filamentous phage expression, secretion and/or display of the compositions or directed mutagens thereof. Certain exemplary polynucleotides are disclosed herein, however, other polynucleotides which, given the degeneracy of the genetic code or codon preferences in a given expression system, encode the FN3 domains of the invention are also within the scope of the invention.

The invention also provides an isolated polynucleotide encoding the FN3 domain specifically binding CD137 comprising the amino acid sequence of SEQ ID NOs: 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222, 223, or 224.

The polynucleotides of the invention may be produced by chemical synthesis such as solid phase polynucleotide synthesis on an automated polynucleotide synthesizer and assembled into complete single or double stranded molecules. Alternatively, the polynucleotides of the invention may be produced by other techniques such as PCR followed by routine cloning. Techniques for producing or obtaining polynucleotides of a given known sequence are well known in the art.

The polynucleotides of the invention may comprise at least one non-coding sequence, such as a promoter or enhancer sequence, intron, polyadenylation signal, a cis sequence facilitating RepA binding, and the like. The polynucleotide sequences may also comprise additional sequences encoding additional amino acids that encode for example a marker or a tag sequence such as a histidine tag or an HA tag to facilitate purification or detection of the protein, a signal sequence, a fusion protein partner such as RepA, Fc or bacteriophage coat protein such as pIX or pIII.

The invention also provides a vector comprising at least one polynucleotide of the invention. Such vectors may be plasmid vectors, viral vectors, vectors for baculovirus expression, transposon based vectors or any other vector suitable for introduction of the polynucleotides of the invention into a given organism or genetic background by any means. Such vectors may be expression vectors comprising nucleic acid sequence elements that can control, regulate, cause or permit expression of a polypeptide encoded by such a vector. Such elements may comprise transcriptional enhancer binding sites, RNA polymerase initiation sites, ribosome binding sites, and other sites that facilitate the expression of encoded polypeptides in a given expression system. Such expression systems may be cell-based, or cell-free systems well known in the art.

The invention also provides a host cell comprising the vector of the invention. The FN3 domain that specifically bind CD137 may be optionally produced by a cell line, a mixed cell line, an immortalized cell or clonal population of immortalized cells, as well known in the art. See, e.g., Ausubel, et al., ed., Current Protocols in Molecular Biology, John Wiley & Sons, Inc., NY, NY (1987-2001); Sambrook, et al., Molecular Cloning: A Laboratory Manual, 2^(nd) Edition, Cold Spring Harbor, N.Y. (1989); Harlow and Lane, Antibodies, a Laboratory Manual, Cold Spring Harbor, N.Y. (1989); Colligan, et al., eds., Current Protocols in Immunology, John Wiley & Sons, Inc., NY (1994-2001); Colligan et al., Current Protocols in Protein Science, John Wiley & Sons, NY, NY, (1997-2001).

The host cell chosen for expression may be of mammalian origin or may be selected from COS-1, COS-7, HEK293, BHK21, CHO, BSC-1, He G2, SP2/0, HeLa, myeloma, lymphoma, yeast, insect or plant cells, or any derivative, immortalized or transformed cell thereof. Alternatively, the host cell may be selected from a species or organism incapable of glycosylating polypeptides, e.g. a prokaryotic cell or organism, such as BL21, BL21(DE3), BL21-GOLD(DE3), XL1-Blue, JM109, HMS174, HMS174(DE3), and any of the natural or engineered E. coli spp, Klebsiella spp., or Pseudomonas spp strains.

The invention also provides a method of producing the isolated FN3 domain that specifically binds CD137, comprising culturing the isolated host cell of the invention under conditions such that the isolated FN3 domain that specifically binds CD137 is expressed, and purifying the FN3 domain.

The FN3 domains that specifically bind CD137 may be purified from recombinant cell cultures by well-known methods, for example by protein A purification, ammonium sulfate or ethanol precipitation, acid extraction, anion or cation exchange chromatography, phosphocellulose chromatography, hydrophobic interaction chromatography, affinity chromatography, hydroxylapatite chromatography and lectin chromatography, or high performance liquid chromatography (HPLC).

Anti-Idiotypic Antibodies

The present invention also provides an anti-idiotypic antibody binding to the FN3 domain.

The invention also provides an anti-idiotypic antibody that specifically binds the FN3 domain comprising the amino acid sequences of one of SEQ ID NOs: 45-224.

Kits

The invention also provides a kit comprising the FN3 domain that specifically binds CD137.

The kit may be used for therapeutic uses and as a diagnostic kit.

In some embodiments, the kit comprises the FN3 domain that specifically binds CD137 and reagents for detecting the FN3 domain. The kit can include one or more other elements including: instructions for use; other reagents, e.g., a label, an agent useful for chelating, or otherwise coupling, a radioprotective composition; devices or other materials for preparing the FN3 domain that specifically binds CD137 for administration for imaging, diagnostic or therapeutic purpose; pharmaceutically acceptable carriers; and devices or other materials for administration to a subject.

In some embodiments, the kit comprises the FN3 domain that specifically binds CD137 comprising the amino acid sequences of one of SEQ ID NOs: 45-224.

Uses of CD137 Binding FN3 Domains of the Invention

The FN3 domains that specifically bind CD137 may be used to diagnose, monitor, modulate, treat, alleviate, help prevent the incidence of, or reduce the symptoms of human disease or specific pathologies in cells, tissues, organs, fluid, or, generally, a host. The FN3 domains that specifically bind CD137 may also be used in imaging CD137 positive tumor tissue in a subject. The methods of the invention may be used with an animal patient belonging to any classification. Examples of such animals include mammals such as humans, rodents, dogs, cats and farm animals.

The invention provides a method of diagnosing a subject having, or who is likely to develop cancer of a tissue based on the expression of CD137 by cells of the cancer tissue, methods of predicting success of immunotherapy, methods of prognosis, and methods of treatment.

The invention also provides a method of detecting CD137-expressing cancer cells in a tumor tissue, comprising

obtaining a sample of the tumor tissue from a subject;

detecting whether CD137 is expressed in the tumor tissue by contacting toe sample of the tumor tissues with the FN3 domain that specifically binds CD137 comprising the amino acid sequence of one of SEQ ID NOs: 45-224 and detecting the binding between CD137 and the FN3 domain.

The tissue can be tissue of any organ or anatomical system, for example lung, epithelial, connective, vascular, muscle, neural, skeletal, lymphatic, prostate, cervical, breast, spleen, gastric, intestinal, oral, esophageal, uterine, ovarian, renal or testicular tissue.

CD137 expression may be evaluated using known methods, such as immunohistochemistry or ELISA.

The invention also provides a method of isolating CD137 expressing cells, comprising

obtaining a sample from a subject;

contacting the sample with the FN3 domain that specifically binds CD137 comprising the amino acid sequence of one of SEQ ID NOs: 45-224, and

isolating the cells bound to the FN3 domains.

The invention also provides a method of detecting CD137-expressing cancer cells in a tumor tissue, comprising

-   -   conjugating the FN3 domain that specifically binds CD137         comprising the amino acid sequence of one of SEQ ID NOs: 45-224         to a detectable label to form a conjugate;     -   administering the conjugate to a subject; and     -   visualizing the CD137 expressing cancer cells to which the         conjugate is bound.

The invention also provides a method of treating a subject having cancer, comprising administering to the subject a FN3 domain that specifically binds CD137 of the invention.

In some embodiments, the subject has a solid tumor.

In some embodiments, the subject has a hematological malignancy.

In some embodiments, the solid tumor is a melanoma.

In some embodiments, the solid tumor is a lung cancer.

In some embodiments, the solid tumor is a non-small cell lung cancer (NSCLC).

In some embodiments, the solid tumor is a squamous non-small cell lung cancer (NSCLC).

In some embodiments, the solid tumor is a non-squamous NSCLC.

In some embodiments, the solid tumor is a lung adenocarcinoma.

In some embodiments, the solid tumor is a renal cell carcinoma (RCC).

In some embodiments, the solid tumor is a mesothelioma.

In some embodiments, the solid tumor is a nasopharyngeal carcinoma (NPC).

In some embodiments, the solid tumor is a colorectal cancer.

In some embodiments, the solid tumor is a prostate cancer.

In some embodiments, the solid tumor is castration-resistant prostate cancer.

In some embodiments, the solid tumor is a stomach cancer.

In some embodiments, the solid tumor is an ovarian cancer.

In some embodiments, the solid tumor is a gastric cancer.

In some embodiments, the solid tumor is a liver cancer.

In some embodiments, the solid tumor is pancreatic cancer.

In some embodiments, the solid tumor is a thyroid cancer.

In some embodiments, the solid tumor is a squamous cell carcinoma of the head and neck.

In some embodiments, the solid tumor is a carcinomas of the esophagus or gastrointestinal tract.

In some embodiments, the solid tumor is a breast cancer.

In some embodiments, the solid tumor is a fallopian tube cancer.

In some embodiments, the solid tumor is a brain cancer.

In some embodiments, the solid tumor is an urethral cancer.

In some embodiments, the solid tumor is a genitourinary cancer.

In some embodiments, the solid tumor is an endometriosis.

In some embodiments, the solid tumor is a cervical cancer.

In some embodiments, the solid tumor is a metastatic lesion of the cancer.

In some embodiments, the hematological malignancy is a lymphoma, a myeloma or a leukemia.

In some embodiments, the hematological malignancy is a B cell lymphoma.

In some embodiments, the hematological malignancy is Burkitt's lymphoma.

In some embodiments, the hematological malignancy is Hodgkin's lymphoma.

In some embodiments, the hematological malignancy is a non-Hodgkin's lymphoma.

In some embodiments, the hematological malignancy is a myelodysplastic syndrome.

In some embodiments, the hematological malignancy is an acute myeloid leukemia (AML).

In some embodiments, the hematological malignancy is a chronic myeloid leukemia (CML).

In some embodiments, the hematological malignancy is a chronic myelomoncytic leukemia (CMML).

In some embodiments, the hematological malignancy is a multiple myeloma (MM).

In some embodiments, the hematological malignancy is a plasmacytoma. In some embodiments, the cancer is kidney cancer.

“Treat” or “treatment” refers to both therapeutic treatment and prophylactic or preventative measures, wherein the object is to prevent or slow down (lessen) an undesired physiological change or disorder, such as the development or spread of cancer. For purposes of this invention, beneficial or desired clinical results include, but are not limited to, alleviation of symptoms, diminishment of extent of disease, stabilized (i.e., not worsening) state of disease, delay or slowing of disease progression, amelioration or palliation of the disease state, and remission (whether partial or total), whether detectable or undetectable. “Treatment” can also mean prolonging survival as compared to expected survival if not receiving treatment. Those in need of treatment include those already with the condition or disorder as well as those prone to have the condition or disorder or those in which the condition or disorder is to be prevented.

A “therapeutically effective amount” refers to an amount effective, at dosages and for periods of time necessary, to achieve a desired therapeutic result. A therapeutically effective amount of the FN3 domains that specifically bind CD137 of the invention may vary according to factors such as the disease state, age, sex, and weight of the individual. Exemplary indicators of an effective FN3 domain that specifically binds CD137 is improved well-being of the patient, decrease or shrinkage of the size of a tumor, arrested or slowed growth of a tumor, and/or absence of metastasis of cancer cells to other locations in the body.

Administration/Pharmaceutical Compositions

The invention provides for pharmaceutical compositions of the FN3 domains that specifically bind CD137, optionally conjugated to a detectable label or a cytotoxic drug of the invention and a pharmaceutically acceptable carrier. For therapeutic use, the FN3 domains that specifically bind CD137 of the invention may be prepared as pharmaceutical compositions containing an effective amount of the domain or molecule as an active ingredient in a pharmaceutically acceptable carrier. “Carrier” refers to a diluent, adjuvant, excipient, or vehicle with which the active compound is administered. Such vehicles can be liquids, such as water and oils, including those of petroleum, animal, vegetable or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil and the like. For example, 0.4% saline and 0.3% glycine can be used. These solutions are sterile and generally free of particulate matter. They may be sterilized by conventional, well-known sterilization techniques (e.g., filtration). The compositions may contain pharmaceutically acceptable auxiliary substances as required to approximate physiological conditions such as pH adjusting and buffering agents, stabilizing, thickening, lubricating and coloring agents, etc. The concentration of the molecules of the invention in such pharmaceutical formulation can vary widely, i.e., from less than about 0.5%, usually at least about 1% to as much as 15 or 20% by weight and will be selected primarily based on required dose, fluid volumes, viscosities, etc., according to the particular mode of administration selected. Suitable vehicles and formulations, inclusive of other human proteins, e.g., human serum albumin, are described, for example, in e.g. Remington: The Science and Practice of Pharmacy, 21^(st) Edition, Troy, D. B. ed., Lipincott Williams and Wilkins, Philadelphia, Pa. 2006, Part 5, Pharmaceutical Manufacturing pp 691-1092, See especially pp. 958-989.

The mode of administration for therapeutic use of the FN3 domains of the invention may be any suitable route that delivers the agent to the host, such as parenteral administration, e.g., intradermal, intramuscular, intraperitoneal, intravenous or subcutaneous, pulmonary; transmucosal (oral, intranasal, intravaginal, rectal), using a formulation in a tablet, capsule, solution, powder, gel, particle; and contained in a syringe, an implanted device, osmotic pump, cartridge, micropump; or other means appreciated by the skilled artisan, as well known in the art. Site specific administration may be achieved by for example intrarticular, intrabronchial, intraabdominal, intracapsular, intracartilaginous, intracavitary, intracelial, intracerebellar, intracerebroventricular, intracolic, intracervical, intragastric, intrahepatic, intracardial, intraosteal, intrapelvic, intrapericardiac, intraperitoneal, intrapleural, intraprostatic, intrapulmonary, intrarectal, intrarenal, intraretinal, intraspinal, intrasynovial, intrathoracic, intrauterine, intravascular, intravesical, intralesional, vaginal, rectal, buccal, sublingual, intranasal, or transdermal delivery.

Pharmaceutical compositions can be supplied as a kit comprising a container that comprises the pharmaceutical composition as described herein. A pharmaceutical composition can be provided, for example, in the form of an injectable solution for single or multiple doses, or as a sterile powder that will be reconstituted before injection. Alternatively, such a kit can include a dry-powder disperser, liquid aerosol generator, or nebulizer for administration of a pharmaceutical composition. Such a kit can further comprise written information on indications and usage of the pharmaceutical composition.

While having described the invention in general terms, the embodiments of the invention will be further disclosed in the following examples that should not be construed as limiting the scope of the claims.

EXAMPLES Example 1: Construction of Tencon Libraries with Randomized Loops

Tencon (SEQ ID NO:1) is an immunoglobulin-like scaffold, fibronectin type III (FN3) domain, designed from a consensus sequence of fifteen FN3 domains from human tenascin-C (Jacobs et al., Protein Engineering, Design, and Selection, 25:107-117, 2012; U.S. Pat. No. 8,278,419). The crystal structure of Tencon shows six surface-exposed loops that connect seven beta-strands. These loops, or selected residues within each loop, can be randomized in order to construct libraries of fibronectin type III (FN3) domains that can be used to select novel molecules that bind to specific targets.

Tencon: (SEQ ID NO 1): LPAPKNLVVSEVTEDSLRLSWTAPDAAFDSFLIQYQESEKVGEAI NLTVPGSERSYDLTGLKPGTEYTVSIYGVKGGHRSNPLSAEFTT Various libraries were generated using the tencon scaffold and various design strategies. In general, libraries TCL1 and TCL2 produced good binders. Generation of TCL1 and TCL2 libraries are described in detail in Int. Pat. Publ. No. WO/2014081944A2. Construction of TCL1 Library

A library designed to randomize only the FG loop of Tencon (SEQ ID NO:1), TCL1, was constructed for use with the cis-display system (Jacobs et al., Protein Engineering, Design, and Selection, 25:107-117, 2012). In this system, a single-strand DNA incorporating sequences for a Tac promoter, Tencon library coding sequence, RepA coding sequence, cis-element, and ori element is produced. Upon expression in an in vitro transcription/translation system, a complex is produced of the Tencon-RepA fusion protein bound in cis to the DNA from which it is encoded. Complexes that bind to a target molecule are then isolated and amplified by polymerase chain reaction (PCR), as described below.

Construction of the TCL1 library for use with cis-display was achieved by successive rounds of PCR to produce the final linear, double-stranded DNA molecules in two halves; the 5′ fragment contains the promoter and Tencon sequences, while the 3′ fragment contains the repA gene and the cis- and ori elements. These two halves are combined by restriction digest in order to produce the entire construct. The TCL1 library was designed to incorporate random amino acids only in the FG loop of Tencon, KGGHRSN (SEQ ID NO:55). NNS codons were used in the construction of this library, resulting in the possible incorporation of all 20 amino acids and one stop codon into the FG loop. The TCL1 library contains six separate sub-libraries, each having a different randomized FG loop length, from 7 to 12 residues, in order to further increase diversity.

TCL1 library (SEQ ID NO: 2) LPAPKNLVVSEVTEDSLRLSWTAPDAAFDSFLIQYQESEKVGE AINLTVPGSERSYDLTGLKPGTEYTVSIYGVX₇₋₁₂PLSAEFTT; wherein X₁, X₂, X₃, X₄, X₅, X₆, X₇ is any amino acid; and X₈, X₉, X₁₀, X₁₁ and X₁₂ are any amino acid or deleted Construction of TCL2 Library

TCL2 library was constructed in which both the BC and the FG loops of Tencon were randomized and the distribution of amino acids at each position was strictly controlled. Table 3 shows the amino acid distribution at desired loop positions in the TCL2 library. The designed amino acid distribution had two aims. First, the library was biased toward residues that were predicted to be structurally important for Tencon folding and stability based on analysis of the Tencon crystal structure and/or from homology modeling. For example, position 29 was fixed to be only a subset of hydrophobic amino acids, as this residue was buried in the hydrophobic core of the Tencon fold. A second layer of design included biasing the amino acid distribution toward that of residues preferentially found in the heavy chain HCDR3 of antibodies, to efficiently produce high-affinity binders (Birtalan et al., J Mol Biol 377:1518-28, 2008; Olson et al., Protein Sci 16:476-84, 2007). Towards this goal, the “designed distribution” in Table 2 refers to the distribution as follows: 6% alanine, 6% arginine, 3.9% asparagine, 7.5% aspartic acid, 2.5% glutamic acid, 1.5% glutamine, 15% glycine, 2.3% histidine, 2.5% isoleucine, 5% leucine, 1.5% lysine, 2.5% phenylalanine, 4% proline, 10% serine, 4.5% threonine, 4% tryptophan, 17.3% tyrosine, and 4% valine. This distribution is devoid of methionine, cysteine, and STOP codons.

TCL2 library (SEQ ID NO: 3) LPAPKNLVVSEVTEDSLRLSWX₁X₂X₃X₄X₅X₆X₇X₈SFLIQYQESEK VGEAINLTVPGSERSYDLTGLKPGTEYTVSIYGVX₉X₁₀X₁₁X₁₂X₁₃ SX₁₄X₁₅LSAEFTT; wherein X₁ is Ala, Arg, Asn, Asp, Glu, Gln, Gly, His, Ile, Leu, Lys, Phe, Pro, Ser, Thr, Trp, Tyr or Val; X₂ is Ala, Arg, Asn, Asp, Glu, Gln, Gly, His, Ile, Leu, Lys, Phe, Pro, Ser, Thr, Trp, Tyr or Val; X₃ Ala, Arg, Asn, Asp, Glu, Gln, Gly, His, Ile, Leu, Lys, Phe, Pro, Ser, Thr, Trp, Tyr or Val; X₄ is Ala, Arg, Asn, Asp, Glu, Gln, Gly, His, Ile, Leu, Lys, Phe, Pro, Ser, Thr, Trp, Tyr or Val; X₅ is Ala, Arg, Asn, Asp, Glu, Gln, Gly, His, Ile, Leu, Lys, Phe, Pro, Ser, Thr, Trp, Tyr or Val; X₆ is Ala, Arg, Asn, Asp, Glu, Gln, Gly, His, Ile, Leu, Lys, Phe, Pro, Ser, Thr, Trp, Tyr or Val; X₇ is Phe, Ile, Leu, Val or Tyr; X₈ is Asp, Glu or Thr; X₉ is Ala, Arg, Asn, Asp, Glu, Gln, Gly, His, Ile, Leu, Lys, Phe, Pro, Ser, Thr, Trp, Tyr or Val; X₁₀ is Ala, Arg, Asn, Asp, Glu, Gln, Gly, His, Ile, Leu, Lys, Phe, Pro, Ser, Thr, Trp, Tyr or Val; X₁₁ is Ala, Arg, Asn, Asp, Glu, Gln, Gly, His, Ile, Leu, Lys, Phe, Pro, Ser, Thr, Trp, Tyr or Val; X₁₂ is Ala, Arg, Asn, Asp, Glu, Gln, Gly, His, Ile, Leu, Lys, Phe, Pro, Ser, Thr, Trp, Tyr or Val; X₁₃ is Ala, Arg, Asn, Asp, Glu, Gln, Gly, His, Ile, Leu, Lys, Phe, Pro, Ser, Thr, Trp, Tyr or Val; X₁₄ is Ala, Arg, Asn, Asp, Glu, Gln, Gly, His, Ile, Leu, Lys, Phe, Pro, Ser, Thr, Trp, Tyr or Val; and X₁₅ is Ala, Arg, Asn, Asp, Glu, Gln, Gly, His, Ile, Leu, Lys, Phe, Pro, Ser, Thr, Trp, Tyr or Val.

TABLE 2 Residue distribution in the TCL2 library Residue WT Position* residues Distribution in the TCL2 library 22 T designed distribution 23 A designed distribution 24 P 50% P + designed distribution 25 D designed distribution 26 A 20% A + 20% G + designed distribution 27 A designed distribution 28 F 20% F, 20% I, 20% L, 20% V, 20% Y 29 D 33% D, 33% E, 33% T 75 K designed distribution 76 G designed distribution 77 G designed distribution 78 H designed distribution 79 R designed distribution 80 S 100% S 81 N designed distribution 82 P 50% P + designed distribution *residue numbering is based on Tencon sequence of SEQ ID NO: 1

Subsequently, these libraries were improved by various ways, including building of the libraries on a stabilized Tencon framework (U.S. Pat. No. 8,569,227) that incorporates substitutions E11R/L17A/N46V/E86I (Tencon27; SEQ ID NO:4) when compared to the wild type tencon as well as altering of the positions randomized in the BC and FG loops. Tencon27 is described in Int. Pat. Appl. No. WO2013049275. From this, new libraries designed to randomize only the FG loop of Tencon (library TCL9), or a combination of the BC and FG loops (library TCL7) were generated. These libraries were constructed for use with the cis-display system (Odegrip et al., Proc Natl Acad Sci USA 101: 2806-2810, 2004). The details of this design are shown below:

Stabilized Tencon (Tencon27) (SEQ ID NO: 4) LPAPKNLVVSRVTEDSARLSWTAPDAAFDSFLIQYQESEKVGEAIV LTVPGSERSYDLTGLKPGTEYTVSIYGVKGGHRSNPLSAIFTT TCL7 (randomized FG and BC loops) (SEQ ID NO: 5) LPAPKNLVVSRVTEDSARLSWX₁X₂X₃X₄X₅X₆X₇X₈X₉FDSFLIQYQE SEKVGEAIVLTVPGSERSYDLTGLKPGTEYTVSIYGVX₁₀X₁₁X₁₂X₁₃ X₁₄X₁₅X₁₆X₁₇X₁₈X₁₉SNPLSAIFTT; wherein X₁, X₂, X₃, X₄, X₅, X₆, X₁₀, X₁₁, X₁₂, X₁₃, X₁₄, X₁₅ and X₁₆ is A, D, E, F, G, H, I, K, L, N, P, Q, R, S, T, V, W or Y; and X₇, X₈, X₉, X₁₇, X₁₈ and X₁₉, is A, D, E, F, G, H, I, K, L, N, P, Q, R, S, T, V, W, Y or deleted. TCL9 (randomized FG loop) (SEQ ID NO: 6) LPAPKNLVVSRVTEDSARLSWTAPDAAFDSFLIQYQESEKVGEAIV LTVPGSERSYDLTGLKPGTEYTVSIYGV X₁X₂X₃X₄X₅X₆X₇X₈X₉ X₁₀X₁₁X₁₂SNPLSAIFTT; X₁, X₂, X₃, X₄, X₅, X₆ and X₇, is A, D, E, F, G, H, I, K, L, N, P, Q, R, S, T, V, W or Y; and X₈, X₉, X₁₀, X₁₁ and X₁₂ is A. D, E, F, G, H, I, K, L, N. P, Q, R, S, T, V, W, Y or deleted.

For library construction, DNA fragments encoding randomized BC loops (lengths 6-9 positions) or FG loops (lengths 7-12 positions) were synthesized using Slonomics technology (Sloning Biotechnology GmbH) so as to control the amino acid distribution of the library and to eliminate stop codons. Two different sets of DNA molecules randomizing either the BC loop or the FG loops were synthesized independently and later combined using PCR to produce the full library product.

Construction of FG Loop Libraries (TCL9)

A set of synthetic DNA molecules consisting of a 5′ Tac promoter followed by the complete gene sequence of Tencon with the exception of randomized codons in the FG loop was produced (SEQ ID NOs: 26-31). For FG loop randomization, all amino acids except cysteine and methionine were encoded at equal percentages. The lengths of the diversified portion are such that they encode for 7, 8, 9, 10, 11, or 12 amino acids in the FG loop. Sub-libraries of each length variation were synthesized individually at a scale of 2 ug and then amplified by PCR using oligos Sloning-FOR (SEQ ID NO:9) and Sloning-Rev (SEQ ID NO:10).

The 3′ fragment of the library is a constant DNA sequence containing elements for display, including a PspOMI restriction site, the coding region of the repA gene, and the cis- and ori elements. PCR reactions were performed to amplify this fragment using a plasmid (pCR4Blunt) (Invitrogen) as a template with M13 Forward and M13 Reverse primers. The resulting PCR products were digested by PspOMI overnight and gel-purified. To ligate the 5′ portion of library DNA to the 3′ DNA containing repA gene, 2 pmol (˜540 ng to 560 ng) of 5′ DNA was ligated to an equal molar (˜1.25 μg) of 3′ repA DNA in the presence of NotI and PspOMI enzyme and T4 ligase at 37° C. overnight. The ligated library product was amplified by using 12 cycles of PCR with oligos POP2250 (SEQ ID NO:11) and DigLigRev (SEQ ID NO:12). For each sub-library, the resulting DNA from 12 PCR reactions were combined and purified by Qiagen spin column. The yield for each sub-library of TCL9 ranged from 32-34 μg.

Construction of FG/BC Loop Libraries (TCL7)

The TCL7 library provides for a library with randomized Tencon BC and FG loops. In this library, BC loops of lengths 6-9 amino acids were mixed combinatorially with randomized FG loops of 7-12 amino acids in length. Synthetic Tencon fragments BC6, BC7, BC8, and BC9 (SEQ ID NOs: 13-16, respectively) were produced to include the Tencon gene encoding for the N-terminal portion of the protein up to and including residue VX such that the BC loop is replaced with either 6, 7, 8, or 9 randomized amino acids. These fragments were synthesized prior to the discovery of L17A, N46V and E83I mutations (CEN5243) but these mutations were introduced in the molecular biology steps described below. In order to combine this fragment with fragments encoding for randomized FG loops, the following steps were taken.

First, a DNA fragment encoding the Tac promoter and the 5′ sequence of Tencon up to the nucleotide encoding for amino acid A17 (130mer-L17A, SEQ ID NO:17) was produced by PCR using oligos POP2222ext (SEQ ID NO:18) and LS1114 (SEQ ID NO:19). This was done to include the L17A mutation in the library (CEN5243). Next, DNA fragments encoding for Tencon residues R18-V75 including randomized BC loops were amplified by PCR using BC6, BC7, BC8, or BC9 as a template and oligos LS1115 (SEQ ID NO:20) and LS1117 (SEQ ID NO:21). This PCR step introduced a BsaI site at the 3′ end. These DNA fragments were subsequently joined by overlapping PCR using oligos POP2222ext and LS1117 as primers. The resulting PCR product of 240 bp was pooled and purified by Qiagen PCR purification kit. The purified DNA was digested with BsaI-HF and gel purified.

Fragments encoding the FG loop were amplified by PCR using FG7, FG8, FG9, FG10, FG11, and FG12 as templates with oligonucleotides SDG10 (SEQ ID NO:22) and SDG24 (SEQ ID NO:23) to incorporate a BsaI restriction site and N46V and E86I variations (CEN5243).

The digested BC fragments and FG fragments were ligated together in a single step using a 3-way ligation. Four ligation reactions in the 16 possible combinations were set up, with each ligation reaction combining two BC loop lengths with 2 FG loop lengths. Each ligation contained ˜300 ng of total BC fragment and 300 ng of the FG fragment. These 4 ligation pools were then amplified by PCR using oligos POP2222 (SEQ ID NO:24) and SDG28 SEQ ID NO:25). 7.5 μg of each reaction product were then digested with NotI and cleaned up with a Qiagen PCR purification column. 5.2 μg of this DNA, was ligated to an equal molar amount of RepA DNA fragment (˜14 μg) digested with PspOMI and the product amplified by PCR using oligos POP2222.

Example 2: Generation of Tencon Libraries Having Alternative Binding Surfaces

The choice of residues to be randomized in a particular library design governs the overall shape of the interaction surface created. X-ray crystallographic analysis of an FN3 domain containing scaffold protein selected to bind maltose binding protein (MBP) from a library in which the BC, DE, and FG loops were randomized was shown to have a largely curved interface that fits into the active site of MBP (Koide et al., Proc Natl Acad Sci USA 104: 6632-6637, 2007). In contrast, an ankyrin repeat scaffold protein that was selected to bind to MBP was found to have a much more planar interaction surface and to bind to the outer surface of MBP distant from the active (Binz et al., Nat Biotechnol 22: 575-582, 2004). These results suggest that the shape of the binding surface of a scaffold molecule (curved vs. flat) may dictate what target proteins or specific epitopes on those target proteins are able to be bound effectively by the scaffold. Published efforts around engineering protein scaffolds containing FN3 domains for protein binding has relied on engineering adjacent loops for target binding, thus producing curved binding surfaces. This approach may limit the number of targets and epitopes accessible by such scaffolds.

Tencon and other FN3 domains contain two sets of CDR-like loops lying on the opposite faces of the molecule, the first set formed by the BC, DE, and FG loops, and the second set formed by the AB, CD, and EF loops. The two sets of loops are separated by the beta-strands that form the center of the FN3 structure. If the image of the Tencon is rotated by 90 degrees, an alternative surface can be visualized. This slightly concave surface is formed by the CD and FG loops and two antiparallel beta-strands, the C and the F beta-strands, and is herein called the C-CD-F-FG surface. The C-CD-F-FG surface can be used as a template to design libraries of protein scaffold interaction surfaces by randomizing a subset of residues that form the surface. Beta-strands have a repeating structure with the side chain of every other residue exposed to the surface of the protein. Thus, a library can be made by randomizing some or all surface exposed residues in the beta strands. By choosing the appropriate residues in the beta-strands, the inherent stability of the Tencon scaffold should be minimally compromised while providing a unique scaffold surface for interaction with other proteins.

Library TCL14 (SEQ ID NO:7), was designed into Tencon27 scaffold (SEQ ID NO:4).

A full description of the methods used to construct this library is described in US. Pat. Publ. No. 2013/0226834.

TCL14 library (SEQ ID NO: 7): LPAPKNLVVSRVTEDSARLSWTAPDAAFDSFX₁IX₂YX₃EX₄X₅ X₆X₇GEAIVLTVPGSERSYDLTGLKPGTEYX₈VX₉IX₁₀GVKGG X₁₁X₁₂SX₁₃PLSAIFTT; wherein X₁, X₂, X₃, X₄, X₅, X₆, X₇, X₈, X₉, X₁₀, X₁₁, X₁₂ and X₁₃ are A, D, E, F, G, H, I, K, L, N, P, Q, R, S, T, V, W, Y, C or M.

The two beta strands forming the C-CD-F-FG surface in Tencon27 have a total of 9 surface exposed residues that could be randomized; C-strand: S30, L32, Q34, Q36; F-strand: E66, T68, S70, Y72, and V74, while the CD loop has 6 potential residues: S38, E39, K40, V41, G42, and E43 and the FG loop has 7 potential residues: K75, G76, G77, H78, R79, S80, and N81. Select residues were chosen for inclusion in the TCL14 design due to the larger theoretical size of the library if all 22 residues were randomized.

Thirteen positions in Tencon were chosen for randomizing: L32, Q34 and Q36 in C-strand, S38, E39, K40 and V41 in CD-loop, T68, S70 and Y72 in F-strand, H78, R79, and N81 in FG-loop. In the C and F strands S30 and E66 were not randomized as they lie just beyond the CD and FG loops and do not appear to be as apparently a part of the C-CD-F-FG surface. For the CD loop, G42 and E43 were not randomized as glycine, providing flexibility, can be valuable in loop regions, and E43 lies at the junction of the surface. The FG loop had K75, G76, G77, and S80 excluded. The glycines were excluded for the reasons above while careful inspection of the crystal structures revealed S80 making key contacts with the core to help form the stable FG loop. K75 faces away from the surface of the C-CD-F-FG surface and was a less appealing candidate for randomization. Although the above mentioned residues were not randomized in the original TCL14 design, they could be included in subsequent library designs to provide additional diversity for de novo selection or for example for an affinity maturation library on a select TCL14 target specific hit.

Subsequent to the production of TCL14, 3 additional Tencon libraries of similar design were produced. These two libraries, TCL19, TCL21 and TCL23, are randomized at the same positions as TCL14 (see above) however the distribution of amino acids occurring at these positions is altered (Table 3). TCL19 and TCL21 were designed to include an equal distribution of 18 natural amino acids at every position (5.55% of each), excluding only cysteine and methionine. TCL23 was designed such that each randomized position approximates the amino acid distribution found in the HCDR3 loops of functional antibodies (Birtalan et al., J Mol Biol 377: 1518-1528, 2008) as described in Table 3. As with the TCL21 library, cysteine and methionine were excluded.

A third additional library was built to expand potential target binding surface of the other libraries library. In this library, TCL24, 4 additional Tencon positions were randomized as compared to libraries TCL14, TCL19, TCL21, and TCL23. These positions include N46 and T48 from the D strand and S84 and 186 from the G strand. Positions 46, 48, 84, and 86 were chosen in particular as the side chains of these residues are surface exposed from beta-strands D and G and lie structurally adjacent to the randomized portions of the C and F strand, thus increasing the surface area accessible for binding to target proteins. The amino acid distribution used at each position for TCL24 is identical to that described for TCL19 and TCL21 in Table 3.

TCL24 Library (SEQ ID NO: 8) LPAPKNLVVSRVTEDSARLSWTAPDAAFDSFX₁IX₂YX₃EX₄X₅X₆ X₇GEAIX₈LX₉VPGSERSYDLTGLKPGTEYX₁₀NX₁₁IX₁₂GVKGG X₁₃X₁₄SX₁₅PLX₁₆AX₁₇FTT; wherein X₁, X₂, X₃, X₄, X₅, X₆, X₁₀, X₁₁, X₁₂, X₁₃, X₁₄, X₁₅, X₁₆ and X₁₇ are A, D, E, F, G, H, I, K, L, N, P, Q, R, S, T, V, Y or W.

TABLE 3 Amino acid frequency (%) at each randomized position for TCL21, TCL23, and TCL24. Amino Acid TCL19 TCL21 TCL23 TCL24 Ala 5.6 5.6 6.0 5.6 Arg 5.6 5.6 6.0 5.6 Asn 5.6 5.6 3.9 5.6 Asp 5.6 5.6 7.5 5.6 Cys 0.0 0.0 0.0 0.0 Gln 5.6 5.6 1.5 5.6 Glu 5.6 5.6 2.5 5.6 Gly 5.6 5.6 15.0 5.6 His 5.6 5.6 2.3 5.6 Ile 5.6 5.6 2.5 5.6 Leu 5.6 5.6 5.0 5.6 Lys 5.6 5.6 1.5 5.6 Met 0.0 0.0 0.0 0.0 Phe 5.6 5.6 2.5 5.6 Pro 5.6 5.6 4.0 5.6 Ser 5.6 5.6 10.0 5.6 Thr 5.6 5.6 4.5 5.6 Trp 5.6 5.6 4.0 5.6 Tyr 5.6 5.6 17.3 5.6 Val 5.6 5.6 4.0 5.6 Generation of TCL21, TCL23, and TCL24 Libraries

The TCL21 library was generated using Colibra library technology (Isogenica) in order to control amino acid distributions. TCL19, TCL23, and TCL24 gene fragments were generated using Slonomics technology (Morphosys) to control amino acid distributions. PCR was used to amplify each library following initial synthesis followed by ligation to the gene for RepA in order to be used in selections using the CIS-display system (Odegrip et al., Proc Natl Acad Sci USA 101: 2806-2810, 2004) as described above for the loop libraries.

Example 3: Selection of Fibronectin Type III (FN3) Domains that Bind CD137

Panning

FN3 domains specific for human CD137 were selected via CIS-Display (Odegrip et al 2004) using recombinant biotinylated CD137 protein (Fc-fusion protein, R&D Systems 838-4B). For in vitro transcription and translation (ITT), 3 μg of DNA from Centyrin libraries TCL18, TCL19, TCL21, TCL23, and TCL24 (See accompanying library description document) were incubated at 30° C. with 0.1 mM complete amino acids, 1× S30 premix components, and 15 μL of S30 extract (Isogenica) in a total volume of 50 pt. After 1 hour, 375 μL of blocking solution (2% BSA in PBS, Invitrogen) was added and reactions were incubated on a cold block for 15 minutes. Unbound library members were removed by successive washes with TBST and TBS. After washing, DNA was eluted from the target protein by heating to 75° C. for 10 minutes and amplified by PCR using KOD polymberase for further rounds of panning. High affinity binders were isolated by successively lowering the concentration of target CD137 during each round from 400 nM to 100 nM and increasing the washing stringency.

Outputs from the fifth round panning were subjected to four additional rounds of off-rate selection. Library transcription and translation was performed as described above after which, the ITT reactions were incubated with biotinylated recombinant CD137 proteins and captured on neutravidin or streptavidin coated magnetic beads, before being washed in TBST extensively then subsequently washed in 5 uM cold recombinant CD137 protein for 1 hour. The biotinylated target antigen concentration was reduced from 25 nM in rounds 6 and 7 to 2.5 nM in rounds 8 and 9.

Following panning, genes encoding the selected FN3 domains were amplified by PCR, subcloned into a pET vector modified to include a ligase independent cloning site, and transformed into BL21 (DE3) (Stratagene) cells for soluble expression in E. coli using standard molecular biology techniques. A gene sequence encoding a C-terminal poly-histidine tag was added to each Centyrin to enable purification and detection. Cultures were grown to an optical density of 0.6-0.8 in TB medium supplemented with 100 μg/mL carbenicillin in 1 mL 96-well blocks at 37° C. before the addition of IPTG to 1 mM, at which point the temperature was reduced to 30° C. Cells were harvested approximately 16 hours later by centrifugation and frozen at −20° C. Cell lysis was achieved by incubating each pellet in 0.6 mL of BugBuster® HT lysis buffer (Novagen EMD Biosciences) supplemented with 0.2 mg/mL lysozyme with shaking at room temperature for 30 minutes.

Biochemical Screening for FN3 Domains that Recombinant CD137

Streptavidin-coated Maxisorp plates (Nunc catalog 436110) were blocked for 1 h in Starting Block T20 (Pierce) and then coated with biotinylated CD137 (using same antigen as in panning) or negative controls (an unrelated Fc-fused recombinant protein and human serum albumin) for 1 h. Plates were rinsed with TBST and diluted lysate was applied to plates for 1 h. Following additional rinses, wells were treated with HRP-conjugated anti-Centyrin antibody (PAB25) for 1 h and then assayed with POD (Roche catalog 11582950001). The DNA from Centyrin lysates with signals at least 10-fold ELISA signal above that of Fc and HSA controls were sequenced resulting in 78 (Table 1) and 102 (Table 2) unique, readable Centyrin sequences isolated from Round 5 and Round 9 screening respectively.

High-Throughput Expression of Anti-CD137 FN3 Domains 102 Isolated clones from unique hits identified by biochemical binding ELISA from Round 9 were combined for growth into 96-well block plate; clones grew in 1 mL cultures (LB media supplemented with kanamycin for selection) at 37° C. overnight with shaking. For protein expression in 96-block plates, 1 mL TB media supplemented with kanamycin was inoculated with 50 uL of the overnight culture and grown at 37° C. with continual shaking at 300 rpm until OD₆₀₀=0.6−1. Once the target OD was reached, protein expression was induced with addition of IPTG to 1 mM; plates were transferred to 30° C. (300 rpm) for overnight growth. Overnight cultures were centrifuged to harvest the cells; bacterial pellets were stored at −80° C. until ready for use. Pellets were lysed with BugBuster® HT lysis buffer (Novagen EMD Biosciences) and His-tagged FN3 domains purified from the clarified lysates with His MultiTrap™ HP plates (GE Healthcare) and eluted in buffer containing 20 mM sodium phosphate, 500 mM sodium chloride, and 250 mM imidazole at pH 7.4. Purified samples were exchanged into PBS pH 7.4 for analysis using PD MultiTrap™ G-25 plates (GE Healthcare). Size Exclusion Chromatography Analysis

Size exclusion chromatography was used to determine the aggregation state of anti-CD137 FN3 domains. Aliquots (10 μL) of each purified Centyrin were injected onto a Superdex 75 5/150 column (GE Healthcare) at a flow rate of 0.3 mL/min in a mobile phase of PBS pH 7.4. Elution from the column was monitored by absorbance at 280 nm. Tencon protein was included in each run as a control. Agilent ChemStation software was used to analyse the elution profiles. 46 anti-CD137 FN3 domains demonstrated a retention time between 5.2 and 6.4 minutes and only a single SEC peak indicative of monomeric protein.

TABLE 4 Summary of Round 5 Screening Hits ELISA ELISA Fc ELISA SEQ CD137-Fc Control HSA ID FN3 Domain (RLU) (RLU) (RLU) No. ISOP120AR5P1D2 907520 640 640 45 ISOP120AR5P1C3 927040 320 240 46 ISOP120AR5P1H3 769280 400 480 47 ISOP120AR5P1C4 708240 320 240 48 ISOP120AR5P1B5 500640 400 320 49 ISOP120AR5P1C5 425120 320 160 50 ISOP120AR5P1G7 568000 560 480 51 ISOP120AR5P1G8 541200 320 320 52 ISOP120AR5P1D9 636320 320 320 53 ISOP120AR5P1F11 714800 480 320 54 ISOP120AR5P1B12 864240 400 480 55 ISOP120BR5P1C1 437680 480 480 56 ISOP120BR5P1F1 541920 480 480 57 ISOP120BR5P1D2 360800 720 240 58 ISOP120BR5P1E2 882480 5680 4960 59 ISOP120BR5P1F2 298800 400 240 60 ISOP120BR5P1D3 1138560 240 400 61 ISOP120BR5P1H3 874560 2000 720 62 ISOP120BR5P1E4 942320 320 560 63 ISOP120BR5P1G4 580240 480 400 64 ISOP120BR5P1A5 503040 640 400 65 ISOP120BR5P1E6 779120 320 400 66 ISOP120BR5P1B7 564560 400 480 67 ISOP120BR5P1C7 306240 880 240 68 ISOP120BR5P1D8 941680 480 320 69 ISOP120BR5P1E8 906160 480 640 70 ISOP120BR5P1B9 358000 560 400 71 ISOP120BR5P1C9 1272800 320 560 72 ISOP120BR5P1D9 1224720 560 560 73 ISOP120BR5P1A10 573280 36160 26560 74 ISOP120BR5P1G11 485440 480 560 75 ISOP120GR5P1E1 1022960 320 320 76 ISOP120GR5P1G3 1335760 320 320 77 ISOP120GR5P1F5 1283680 400 400 78 ISOP120GR5P1H6 721440 400 400 79 ISOP120GR5P1E7 1130720 400 480 80 ISOP120GR5P1A10 626640 400 400 81 ISOP120GR5P1C10 501840 240 480 82 ISOP120GR5P1A11 1045760 480 320 83 ISOP120GR5P1B11 875360 320 160 84 ISOP120GR5P1H11 1310560 640 320 85 ISOP120HR5P1E2 1319040 720 2880 86 ISOP120HR5P1A3 1076480 560 240 87 ISOP120HR5P1B4 1185360 320 320 88 ISOP120HR5P1G4 346880 320 480 89 ISOP120HR5P1H4 630480 480 320 90 ISOP120HR5P1B5 519520 320 240 91 ISOP120HR5P1A6 1292720 640 400 92 ISOP120HR5P1G6 2035360 400 320 93 ISOP120HR5P1A7 986800 400 480 94 ISOP120HR5P1D7 1104240 320 320 95 ISOP120HR5P1E7 363120 480 480 96 ISOP120HR5P1H7 1527200 640 480 97 ISOP120HR5P1H8 2217040 320 400 98 ISOP120HR5P1D9 404720 480 400 99 ISOP120HR5P1F9 1177120 400 400 100 ISOP120ER5P1B4 499360 400 400 101 ISOP120ER5P1F4 536720 320 400 102 ISOP120ER5P1H4 1070240 480 560 103 ISOP120ER5P1E5 413120 240 320 104 ISOP120ER5P1B6 1351600 160 400 105 ISOP120ER5P1C6 495360 320 400 106 ISOP120ER5P1H6 588560 320 480 107 ISOP120ER5P1A7 1114080 400 400 108 ISOP120ER5P1A8 1897040 400 320 109 ISOP120ER5P1E10 810320 720 400 110 ISOP120ER5P1A11 1144160 320 320 111 ISOP120ER5P1B12 1441520 720 800 112 ISOP120FR5P1F1 1228320 480 640 113 ISOP120FR5P1C2 388960 240 400 114 ISOP120FR5P1H5 459680 400 560 115 ISOP120FR5P1A6 1404240 400 320 116 ISOP120FR5P1H6 356880 320 320 117 ISOP120FR5P1D7 1178800 400 480 118 ISOP120FR5P1F8 1197120 240 400 119 ISOP120FR5P1E9 1183360 320 400 120 ISOP120FR5P1E10 953040 240 320 121 ISOP120FR5P1A11 920080 480 480 122

TABLE 5 Summary of Round 9 Screening Hits ELISA SEC SEC ELISA Fc ELISA Retention Peak CD137 Control HSA Time Height SEQ ID FN3 Domain Clone (RLU) (RLU) (RLU) (min) (mAU) Monomeric No. ISOP193AR9P1A11 8659280 1440 320 No peak FALSE 123 ISOP193AR9P1A6 6739840 960 480 No peak FALSE 124 ISOP193AR9P1B10 8120400 1520 480 5.355 84.76 TRUE 125 ISOP193AR9P1B12 2762240 2240 1440 5.712 39.72 TRUE 126 ISOP193AR9P1B4 5744400 960 480 5.495 94.79 TRUE 127 ISOP193AR9P1C10 7143200 3520 960 No peak FALSE 128 ISOP193AR9P1E6 4179680 1200 720 No peak FALSE 129 ISOP193AR9P1F4 3836000 1520 720 5.664 80.79 TRUE 130 ISOP193AR9P1F9 4710240 1600 560 5.317 142.48 TRUE 131 ISOP193AR9P1G11 5892800 2240 960 5.234 225.17 132 ISOP193AR9P1G5 4022880 1120 720 5.315 191.85 FALSE 133 ISOP193AR9P1G8 4255040 1440 720 No peak 1.40 FALSE 134 ISOP193AR9P1H8 3716320 3040 1040 5.982 36.60 TRUE 135 ISOP193BR9P1B10 9733920 2640 1200 5.884 66.69 TRUE 136 ISOP193BR9P1B12 6551440 14000 2880 No peak FALSE 137 ISOP193BR9P1E6 4625840 2560 560 6.326 5.13 FALSE 138 ISOP193BR9P1G11 4988080 56880 9600 No peak FALSE 139 ISOP193BR9P1G2 6145520 15920 2160 No peak FALSE 140 ISOP193BR9P1G3 4710400 1360 640 7.974 7.68 FALSE 141 ISOP193BR9P1G6 8092720 2640 960 6.045 11.68 TRUE 142 ISOP193BR9P1G9 3725520 1200 720 6.028 4.71 FALSE 143 ISOP193BR9P1H2 3502960 11840 3280 6.055 55.87 FALSE 144 ISOP193BR9P1H3 5257440 2080 1360 No peak FALSE 145 ISOP193BR9P1H6 8857840 8320 2880 5.787 64.85 FALSE 146 ISOP193ER9P1A10 14863840 1120 240 No peak FALSE 147 ISOP193ER9P1A11 12781600 1200 640 6.015 35.38 TRUE 148 ISOP193ER9P1A3 14185440 3040 880 5.73  136.02 TRUE 149 ISOP193ER9P1A4 9806400 960 480 5.738 61.50 TRUE 150 ISOP193ER9P1A8 14274800 1440 400 5.892 14.52 TRUE 151 ISOP193ER9P1B4 16089360 1600 320 No peak FALSE 152 ISOP193ER9P1B5 12675520 1200 400 6.033 8.43 TRUE 153 ISOP193ER9P1C10 8866800 2480 560 5.704 179.28 TRUE 154 ISOP193ER9P1C4 15455120 960 320 6.032 18.18 TRUE 155 ISOP193ER9P1C8 16680560 1040 400 5.862 19.39 TRUE 156 ISOP193ER9P1C9 14280160 880 560 5.668 20.02 TRUE 157 ISOP193ER9P1D4 16022720 1120 480 5.843 22.38 TRUE 158 ISOP193ER9P1D7 10954000 1680 400 5.92  80.79 TRUE 159 ISOP193ER9P1E1 14972480 1200 560 5.755 18.97 TRUE 160 ISOP193ER9P1E2 15691600 1040 560 6.296 20.36 TRUE 161 ISOP193ER9P1E4 12645760 1680 480 5.762 118.21 TRUE 162 ISOP193ER9P1E8 16401200 880 480 5.699 16.07 TRUE 163 ISOP193ER9P1F11 11182240 2240 400 No peak FALSE 164 ISOP193ER9P1F7 15148960 1200 480 5.856 7.39 TRUE 165 ISOP193ER9P1F9 14980400 1840 400 7.819 4.92 FALSE 166 ISOP193ER9P1G11 14840160 1440 560 5.859 17.01 TRUE 167 ISOP193ER9P1G2 7192960 1680 720 5.677 24.15 TRUE 168 ISOP193ER9P1G4 13819760 1440 320 5.979 2.54 FALSE 169 ISOP193ER9P1G5 15073600 1440 400 No peak FALSE 170 ISOP193ER9P1G9 12900320 1280 400 5.781 6.70 TRUE 171 ISOP193ER9P1H11 2080000 1360 640 5.991 39.63 TRUE 172 ISOP193ER9P1H2 5183360 1360 560 5.833 53.11 TRUE 173 ISOP193ER9P1H3 10515760 1520 400 6.073 7.45 FALSE 174 ISOP193FR9P1A11 5784000 3520 880 No peak FALSE 175 ISOP193FR9P1A5 9072080 95120 26240 6.033 8.14 TRUE 176 ISOP193FR9P1C1 14116720 36800 8160 5.866 3.99 FALSE 177 ISOP193FR9P1C5 8660800 79280 19200 6.377 20.91 TRUE 178 ISOP193FR9P1C9 12306480 21040 4000 7.257 5.36 FALSE 179 ISOP193FR9P1D1 8132800 1680 640 No peak FALSE 180 ISOP193FR9P1D5 6046880 51680 10800 5.948 4.84 TRUE 181 ISOP193FR9P1D7 2195360 15040 2640 6.077 1.98 FALSE 182 ISOP193FR9P1E1 11602480 2000 880 5.84  28.48 FALSE 183 ISOP193FR9P1E10 2051600 133120 31040 5.9  46.84 TRUE 184 ISOP193FR9P1F8 8573040 25680 5040 5.652 3.10 FALSE 185 ISOP193FR9P1G10 8908880 2480 880 6.864 2.25 FALSE 186 ISOP193FR9P1G11 10788560 60640 10960 5.945 5.46 TRUE 187 ISOP193FR9P1G2 7864240 2560 880 No peak FALSE 188 ISOP193FR9P1G4 13950480 1840 640 5.834 5.47 TRUE 189 ISOP193FR9P1G7 5500720 42320 10960 5.897 9.89 TRUE 190 ISOP193FR9P1G8 14458880 25120 5040 5.874 5.87 FALSE 191 ISOP193FR9P1G9 12761120 33600 6800 6.413 6.02 FALSE 192 ISOP193FR9P1H6 11204000 88320 23040 5.712 7.91 TRUE 193 ISOP193FR9P1H9 2420400 2000 800 5.987 23.27 TRUE 194 ISOP193GR9P1A7 2153840 1040 480 5.634 10.89 TRUE 195 ISOP193GR9P1B3 3457040 880 320 5.768 3.78 FALSE 196 ISOP193GR9P1E10 10452960 1360 480 No peak FALSE 197 ISOP193GR9P1F6 9846640 1360 400 5.656 4.72 FALSE 198 ISOP193GR9P1F7 3480640 880 400 5.712 2.95 FALSE 199 ISOP193GR9P1G9 3052480 960 480 5.645 7.32 TRUE 200 ISOP193GR9P1H2 5314000 1360 640 No peak FALSE 201 ISOP193HR9P1A10 12663280 5680 1520 No peak FALSE 202 ISOP193HR9P1A11 16644800 28320 4240 No peak FALSE 203 ISOP193HR9P1A5 14895120 6080 2080 No peak FALSE 204 ISOP193HR9P1A6 14635040 24960 5120 No peak FALSE 205 ISOP193HR9P1A7 14786080 48880 12640 6.013 9.21 TRUE 206 ISOP193HR9P1B11 16579440 14960 4080 No peak FALSE 207 ISOP193HR9P1B7 16384560 12960 2240 No peak FALSE 208 ISOP193HR9P1C7 3436800 71360 10000 5.69  2.15 FALSE 209 ISOP193HR9P1C8 18185520 1360 560 6.475 4.54 TRUE 210 ISOP193HR9P1D11 14160720 48720 6240 5.936 5.80 TRUE 211 ISOP193HR9P1D8 6271280 10880 2640 5.79  4.93 TRUE 212 ISOP193HR9P1E2 9022400 13120 3840 5.801 4.22 FALSE 213 ISOP193HR9P1E3 17767600 1120 640 6.564 3.11 FALSE 214 ISOP193HR9P1E6 11258560 20080 3040 5.859 3.13 FALSE 215 ISOP193HR9P1E8 16318560 3120 1520 No peak FALSE 216 ISOP193HR9P1F10 15810240 1280 960 No peak FALSE 217 ISOP193HR9P1F8 16086000 31280 6080 No peak FALSE 218 ISOP193HR9P1G10 15586960 1360 800 6.226 2.97 FALSE 219 ISOP193HR9P1G4 17180000 960 560 6.293 2.54 FALSE 220 ISOP193HR9P1G5 15137440 24160 3360 5.913 9.58 TRUE 221 ISOP193HR9P1G6 11499680 8160 1200 No peak FALSE 222 ISOP193HR9P1H10 14818080 43920 10080 6.107 2.19 FALSE 223 ISOP193HR9P1H7 4604800 49840 13680 6.035 1.81 FALSE 224

Example 4: Characterization of Fibronectin Type III (FN3) Domains that Bind CD137

Fluorescence-Activated Cell Sorting (FACS)

Cell surface binding was analyzed via flow cytometry. Cenyrins were prepared at a maximal concentration of 500 nM in the presence of 125 nM anti His-mIgG1 antibody and then serially diluted in PBS/1% FCS buffer. Samples were then applied to approx 100 000 CHO-Kl cells expressing the extracellular domain of human CD137 on their surface. Unbound Centyrin/Antibody complexes were washed away and bound FN3 domains were detected by the addition of goat anti mouse-FITC labeled antibody. Samples were then aquired by flow cytometry. Subsequently, Mean flurescence intensity was plotted against the log Centyrin concentration and EC50 values were calculated by nonlinear regression using GraphPad Prism.

Binding Analysis with Biacore

For selected FN3 domains, binding to recombinant CD137-Fc was evaluated using surface plasmon resonance (Biacore T100). For each cycle approximately 500 RU of recombinant human CD137-Fc-IgG1 was captured via an anti human IgG immobilized on the surface of a CMS chip. Once CD137-Fc protein was captured, increasing concentrations of Centyrin candidates were injected for 120 s and dissociation was then analyzed for 240 s. A flow cell immobilized with just the anti human IgG1 antibody served as reference flow cell. Kd values were subsequently extrapolated using a 1:1 kinetic binding model (BiaEvaluation software).

TABLE 6 Centyrin binding analysis by FACS and Biacore EC50 FACS Biacore Centyrin Clone (nM) (Kd) ISOP193AR9P1B4 13.93 n.d. ISOP193AR9P1F4 n.a. n.d. ISOP193AR9P1H8 n.a. n.d. ISOP193AR9P1F9 218 n.d. ISOP193AR9P1B10 1.205 n.d. ISOP193AR9P1G11 10.48 n.d. ISOP193AR9P1B12 n.a. n.d. ISOP193BR9P1G6 n.a. n.d. ISOP193BR9P1B10 134 n.d. ISOP193GR9P1A7 605.6 n.d. ISOP193GR9P1G9 n.a. n.d. ISOP193GR9P1F11 508.9 n.d. ISOP193HR9P1C8 3.268 n.d. ISOP193ER9P1E1 12.55 1.059E−08 ISOP193ER9P1E2 15.31 1.094E−08 ISOP193ER9P1G2 22.94 6.563E−08 ISOP193ER9P1H2 27.46 n.d. ISOP193ER9P1A3 10.22  8.3E−09 ISOP193ER9P1A4 27.1 1.172E−07 ISOP193ER9P1C4 18.91  1.31E−08 ISOP193ER9P1D4 12.86 9.552E−09 ISOP193ER9P1E4 278.7 n.d. ISOP193ER9P1B5 7.342 n.d. ISOP193ER9P1D5 29.61 n.d. ISOP193ER9P1C7 19.07 n.d. ISOP193ER9P1F7 24.99 n.d. ISOP193ER9P1A8 16.96 n.d. ISOP193ER9P1C8 12.43 n.d. ISOP193ER9P1E8 17.67 n.d. ISOP193ER9P1C9 14.1  2.51E−08 ISOP193ER9P1G9 15.26 5.986E−08 ISOP193ER9P1C10 31.01 1.077E−07 ISOP193ER9P1A11 10.56 n.d. ISOP193ER9P1G11 18.69 n.d. ISOP193ER9P1H11 184.5 n.d. ISOP193ER9P1B12 19.87 n.d. ISOP193FR9P1G4 26.93 n.d. ISOP193FR9P1H9 72.75 n.d.

SEQUENCES SEQ ID NO: 1 = Original Tencon Sequence LPAPKNLVVSEVTEDSLRLSWTAPDAAFDSFLIQYQESEKVGEAINLTVPGSERSY DLTGLKPGTEYTVSIYGVKGGHRSNPLSAEFTT SEQ ID NO: 2 = TCL1 library LPAPKNLVVSEVTEDSLRLSWTAPDAAFDSFLIQYQESEKVGEAINLTVPGSERSY DLTGLKPGTEYTVSIYGV(X)₇₋₁₂PL5AEFTT; wherein X₁, X₂, X₃, X₄, X₅, X₆, X₇ is any amino acid; and X₈, X₉, X₁₀, X₁₁ and X₁₂ are any amino acid or deleted SEQ ID NO: 3 = TCL2 library LPAPKNLVVSEVTEDSLRLSWX₁X₂X₃X₄X₅X₆X₇X₈SFLIQYQESEKVGEAINLTVPGS ERSYDLTGLKPGTEYTVSIYGVX₉X₁₀X₁₁X₁₂X₁₃SX₁₄X₁₅L5AEFTT; wherein X₁ is Ala, Arg, Asn, Asp, Glu, Gln, Gly, His, Ile, Leu, Lys, Phe, Pro, Ser, Thr, Trp, Tyr or Val; X₂ is Ala, Arg, Asn, Asp, Glu, Gln, Gly, His, Ile, Leu, Lys, Phe, Pro, Ser, Thr, Trp, Tyr or Val; X₃ Ala, Arg, Asn, Asp, Glu, Gln, Gly, His, Ile, Leu, Lys, Phe, Pro, Ser, Thr, Trp, Tyr or Val; X₄ is Ala, Arg, Asn, Asp, Glu, Gln, Gly, His, Ile, Leu, Lys, Phe, Pro, Ser, Thr, Trp, Tyr or Val; X₅ is Ala, Arg, Asn, Asp, Glu, Gln, Gly, His, Ile, Leu, Lys, Phe, Pro, Ser, Thr, Trp, Tyr or Val; X₆ is Ala, Arg, Asn, Asp, Glu, Gln, Gly, His, Ile, Leu, Lys, Phe, Pro, Ser, Thr, Trp, Tyr or Val; X₇ is Phe, Ile, Leu, Val or Tyr; X₈ is Asp, Glu or Thr; X₉ is Ala, Arg, Asn, Asp, Glu, Gln, Gly, His, Ile, Leu, Lys, Phe, Pro, Ser, Thr, Trp, Tyr or Val; X₁₀ is Ala, Arg, Asn, Asp, Glu, Gln, Gly, His, Ile, Leu, Lys, Phe, Pro, Ser, Thr, Trp, Tyr or Val; X₁₁ is Ala, Arg, Asn, Asp, Glu, Gln, Gly, His, Ile, Leu, Lys, Phe, Pro, Ser, Thr, Trp, Tyr or Val; X₁₂ is Ala, Arg, Asn, Asp, Glu, Gln, Gly, His, Ile, Leu, Lys, Phe, Pro, Ser, Thr, Trp, Tyr or Val; X₁₃ is Ala, Arg, Asn, Asp, Glu, Gln, Gly, His, Ile, Leu, Lys, Phe, Pro, Ser, Thr, Trp, Tyr or Val; X₁₄ is Ala, Arg, Asn, Asp, Glu, Gln, Gly, His, Ile, Leu, Lys, Phe, Pro, Ser, Thr, Trp, Tyr or Val; and X₁₅ is Ala, Arg, Asn, Asp, Glu, Gln, Gly, His, Ile, Leu, Lys, Phe, Pro, Ser, Thr, Trp, Tyr or Val. SEQ ID NO: 4 = Stabilized Tencon LPAPKNLVVSRVTEDSARLSWTAPDAAFDSFLIQYQESEKVGEAIVLTVPGSERSY DLTGLKPGTEYTVSIYGVKGGHRSNPLSAIFTT SEQ ID NO: 5 = TCL7 (FG and BC loops) LPAPKNLVVSRVTEDSARLSWX₁X₂X₃X₄X₅X₆X₇X₈X₉FDSFLIQYQESEKVGEAIVLT VPGSERSYDLTGLKPGTEYTVSIYGVX₁₀X₁₁X₁₂X₁₃X₁₄X₁₅X₁₆X₁₇X₁₈X₁₉SNPLSAIFTT; wherein X₁, X₂, X₃, X₄, X₅, X₆, X₁₀, X₁₁, X₁₂, X₁₃, X₁₄, X₁₅ and X₁₆ are A, D, E, F, G, H, I, K, L, N, P, Q, R, S, T, V, W or Y; and X₇, X₈, X₉, X₁₇, X₁₈ and X₁₉, are A, D, E, F, G, H, I, K, L, N, P, Q, R, S, T, V, W, Y or deleted SEQ ID NO: 6 = TCL9 (FG loop) LPAPKNLVVSRVTEDSARLSWTAPDAAFDSFLIQYQESEKVGEAIVLTVPGSERSY DLTGLKPGTEYTVSIYGV X₁X₂X₃X₄X₅X₆X₇X₈X₉X₁₀X₁₁X₁₂SNPLSAIFTT; wherein X₁, X₂, X₃, X₄, X₅, X₆ and X₇, is A, D, E, F, G, H, I, K, L, N, P, Q, R, S, T, V, W or Y; and X₈, X₉, X₁₀, X₁₁ and X₁₂ is A, D, E, F, G, H, I, K, L, N, P, Q, R, S, T, V, W, Y or deleted. TCL14 library (SEQ ID NO: 7): LPAPKNLVVSRVTEDSARLSWTAPDAAFDSFX₁IX₂YX₃EX₄X₅X₆X₇GEAIVLTVPGS ERSYDLTGLKPGTEYX₈VX₉IXioGVKGGX₁₁X₁₂SX₁₃PLSAIFTT; wherein X₁, X₂, X₃, X₄, X₅, X₆, X₇, X₈, X₉, X₁₀, X₁₁, X₁₂ and X₁₃ are A, D, E, F, G, H, I, K, L, N, P, Q, R, S, T, V, W,Y, C or M. TCL24 Library (SEQ ID NO: 8) LPAPKNLVVSRVTEDSARLSWTAPDAAFDSFX₁IX₂YX₃EX₄X₅X₆X₇GEAIX₈LX₉VPG SERSYDLTGLKPGTEYX₁₀VX₁₁IX₁₂GVKGGX₁₃X₁₄SX₁₅PLX₁₆AX₁₇FTT; wherein X₁, X₂, X₃, X₄, X₅, X₆, X₁₀, X₁₁, X₁₂, X₁₃, X₁₄, X₁₅, X₁₆ and X₁₇ are A, D, E, F, G, H, I, K, L, N, P, Q, R, S, T, V, Y or W. SEQ ID NO: 9 = Sloning-FOR GTGACACGGCGGTTAGAAC SEQ ID NO: 10 =  Sloning-REV GCCTTTGGGAAGCTTCTAAG SEQ ID NO: 11 =  POP2250 CGGCGGTTAGAACGCGGCTACAATTAATAC SEQ ID NO: 12 =  DigLigRev CATGATTACGCCAAGCTCAGAA SEQ ID NO: 13 = BC9 GTGACACGGCGGTTAGAACGCGGCTACAATTAATACATAACCCCATCCCCCTG TTGACAATTAATCATCGGCTCGTATAATGTGTGGAATTGTGAGCGGATAACAA TTTCACACAGGAAACAGGATCTACCATGCTGCCGGCGCCGAAAAACCTGGTTG TTTCTGAAGTTACCGAAGACTCTCTGCGTCTGTCTTGGNNNNNNNNNNNNNNN NNNNNNNNNNNNTTYGACTCTTTCCTGATCCAGTACCAGGAATCTGAAAAAGT TGGTGAAGCGATCAACCTGACCGTTCCGGGTTCTGAACGTTCTTACGACCTGA CCGGTCTGAAACCGGGTACCGAATACACCGTTTCTATCTACGGTGTTCTTAGA AGCTTCCCAAAGGC SEQ ID NO: 14 = BC8 GTGACACGGCGGTTAGAACGCGGCTACAATTAATACATAACCCCATCCCCCTG TTGACAATTAATCATCGGCTCGTATAATGTGTGGAATTGTGAGCGGATAACAA TTTCACACAGGAAACAGGATCTACCATGCTGCCGGCGCCGAAAAACCTGGTTG TTTCTGAAGTTACCGAAGACTCTCTGCGTCTGTCTTGGNNNNNNNNNNNNNNN NNNNNNNNNTTYGACTCTTTCCTGATCCAGTACCAGGAATCTGAAAAAGTTGG TGAAGCGATCAACCTGACCGTTCCGGGTTCTGAACGTTCTTACGACCTGACCG GTCTGAAACCGGGTACCGAATACACCGTTTCTATCTACGGTGTTCTTAGAAGC TTCCCAAAGGC SEQ ID NO: 15 = BC7 GTGACACGGCGGTTAGAACGCGGCTACAATTAATACATAACCCCATCCCCCTG TTGACAATTAATCATCGGCTCGTATAATGTGTGGAATTGTGAGCGGATAACAA TTTCACACAGGAAACAGGATCTACCATGCTGCCGGCGCCGAAAAACCTGGTTG TTTCTGAAGTTACCGAAGACTCTCTGCGTCTGTCTTGGNNNNNNNNNNNNNNN NNNNNNTTYGACTCTTTCCTGATCCAGTACCAGGAATCTGAAAAAGTTGGTGA AGCGATCAACCTGACCGTTCCGGGTTCTGAACGTTCTTACGACCTGACCGGTC TGAAACCGGGTACCGAATACACCGTTTCTATCTACGGTGTTCTTAGAAGCTTC CCAAAGGC SEQ ID NO: 16 = BC6 GTGACACGGCGGTTAGAACGCGGCTACAATTAATACATAACCCCATCCCCCTG TTGACAATTAATCATCGGCTCGTATAATGTGTGGAATTGTGAGCGGATAACAA TTTCACACAGGAAACAGGATCTACCATGCTGCCGGCGCCGAAAAACCTGGTTG TTTCTGAAGTTACCGAAGACTCTCTGCGTCTGTCTTGGNNNNNNNNNNNNNNN NNNTTYGACTCTTTCCTGATCCAGTACCAGGAATCTGAAAAAGTTGGTGAAGC GATCAACCTGACCGTTCCGGGTTCTGAACGTTCTTACGACCTGACCGGTCTGA AACCGGGTACCGAATACACCGTTTCTATCTACGGTGTTCTTAGAAGCTTCCCA AAGGCAAGGC SEQ ID NO: 17 = 130mer-L17A CGGCGGTTAGAACGCGGCTACAATTAATACATAACCCCATCCCCCTGTTGACA ATTAATCATCGGCTCGTATAATGTGTGGAATTGTGAGCGGATAACAATTTCAC ACAGGAAACAGGATCTACCATGCTG SEQ ID NO: 18 = POP222ext CGG CGG TTA GAA CGC GGC TAC AAT TAA TAC SEQ ID NO: 19 = LS1114 CCA AGA CAG ACG GGC AGA GTC TTC GGT AAC GCG AGA AAC AAC CAG GTT TTT CGG CGC CGG CAG CAT GGT AGA TCC TGT TTC SEQ ID NO: 20 = LS1115 CCG AAG ACT CTG CCC GTC TGT CTT GG SEQ ID NO: 21 = LS1117 CAG TGG TCT CAC GGA TTC CTG GTA CTG GAT CAG GAA AGA GTC GAA SEQ ID NO: 22 = SDG10 CATGCGGTCTCTTCCGAAAAAGTTGGTGAAGCGATCGTCCTGACCGTTCCGGG T SEQ ID NO: 23 = SDG24 GGTGGTGAAGATCGCAGACAGCGGGTTAG SEQ ID NO: 24 = P0P2222 CGGCGGTTAGAACGCGGCTAC SEQ ID NO: 25 = SDG28 AAGATCAGTTGCGGCCGCTAGACTAGAACCGCTGCCACCGCCGGTGGTGAAG ATCGCAGAC SEQ ID NO: 26 = FG12 GTGACACGGCGGTTAGAACGCGGCTACAATTAATACATAACCCCATCCCCCTG TTGACAATTAATCATCGGCTCGTATAATGTGTGGAATTGTGAGCGGATAACAA TTTCACACAGGAAACAGGATCTACCATGCTGCCGGCGCCGAAAAACCTGGTTG TTTCTCGCGTTACCGAAGACTCTGCGCGTCTGTCTTGGACCGCGCCGGACGCG GCGTTCGACTCTTTCCTGATCCAGTACCAGGAATCTGAAAAAGTTGGTGAAGC GATCGTGCTGACCGTTCCGGGTTCTGAACGTTCTTACGACCTGACCGGTCTGA AACCGGGTACCGAATACACCGTTTCTATCTACGGTGTTNNNNNNNNNNNNNNN NNNNNNNNNNNNNNNNNNNNNTCTAACCCGCTGTCTGCGATCTTCACCACCGG CGGTCACCATCACCATCACCATGGCAGCGGTTCTAGTCTAGCGGCCGCAACTG ATCTTGGC SEQ ID NO: 27 = FG11 GTGACACGGCGGTTAGAACGCGGCTACAATTAATACATAACCCCATCCCCCTG TTGACAATTAATCATCGGCTCGTATAATGTGTGGAATTGTGAGCGGATAACAA TTTCACACAGGAAACAGGATCTACCATGCTGCCGGCGCCGAAAAACCTGGTTG TTTCTCGCGTTACCGAAGACTCTGCGCGTCTGTCTTGGACCGCGCCGGACGCG GCGTTCGACTCTTTCCTGATCCAGTACCAGGAATCTGAAAAAGTTGGTGAAGC GATCGTGCTGACCGTTCCGGGTTCTGAACGTTCTTACGACCTGACCGGTCTGA AACCGGGTACCGAATACACCGTTTCTATCTACGGTGTTNNNNNNNNNNNNNNN NNNNNNNNNNNNNNNNNNTCTAACCCGCTGTCTGCGATCTTCACCACCGGCGG TCACCATCACCATCACCATGGCAGCGGTTCTAGTCTAGCGGCCGCAACTGATC TTGGC SEQ ID NO: 28 = FG10 GTGACACGGCGGTTAGAACGCGGCTACAATTAATACATAACCCCATCCCCCTG TTGACAATTAATCATCGGCTCGTATAATGTGTGGAATTGTGAGCGGATAACAA TTTCACACAGGAAACAGGATCTACCATGCTGCCGGCGCCGAAAAACCTGGTTG TTTCTCGCGTTACCGAAGACTCTGCGCGTCTGTCTTGGACCGCGCCGGACGCG GCGTTCGACTCTTTCCTGATCCAGTACCAGGAATCTGAAAAAGTTGGTGAAGC GATCGTGCTGACCGTTCCGGGTTCTGAACGTTCTTACGACCTGACCGGTCTGA AACCGGGTACCGAATACACCGTTTCTATCTACGGTGTTNNNNNNNNNNNNNNN NNNNNNNNNNNNNNNTCTAACCCGCTGTCTGCGATCTTCACCACCGGCGGTCA CCATCACCATCACCATGGCAGCGGTTCTAGTCTAGCGGCCGCAACTGATCTTG GC SEQ ID NO: 29 = FG9 GTGACACGGCGGTTAGAACGCGGCTACAATTAATACATAACCCCATCCCCCTG TTGACAATTAATCATCGGCTCGTATAATGTGTGGAATTGTGAGCGGATAACAA TTTCACACAGGAAACAGGATCTACCATGCTGCCGGCGCCGAAAAACCTGGTTG TTTCTCGCGTTACCGAAGACTCTGCGCGTCTGTCTTGGACCGCGCCGGACGCG GCGTTCGACTCTTTCCTGATCCAGTACCAGGAATCTGAAAAAGTTGGTGAAGC GATCGTGCTGACCGTTCCGGGTTCTGAACGTTCTTACGACCTGACCGGTCTGA AACCGGGTACCGAATACACCGTTTCTATCTACGGTGTTNNNNNNNNNNNNNNN NNNNNNNNNNNNTCTAACCCGCTGTCTGCGATCTTCACCACCGGCGGTCACCA TCACCATCACCATGGCAGCGGTTCTAGTCTAGCGGCCGCAACTGATCTTGGC SEQ ID NO: 30 = FG8 GTGACACGGCGGTTAGAACGCGGCTACAATTAATACATAACCCCATCCCCCTG TTGACAATTAATCATCGGCTCGTATAATGTGTGGAATTGTGAGCGGATAACAA TTTCACACAGGAAACAGGATCTACCATGCTGCCGGCGCCGAAAAACCTGGTTG TTTCTCGCGTTACCGAAGACTCTGCGCGTCTGTCTTGGACCGCGCCGGACGCG GCGTTCGACTCTTTCCTGATCCAGTACCAGGAATCTGAAAAAGTTGGTGAAGC GATCGTGCTGACCGTTCCGGGTTCTGAACGTTCTTACGACCTGACCGGTCTGA AACCGGGTACCGAATACACCGTTTCTATCTACGGTGTTNNNNNNNNNNNNNNN NNNNNNNNNTCTAACCCGCTGTCTGCGATCTTCACCACCGGCGGTCACCATCA CCATCACCATGGCAGCGGTTCTAGTCTAGCGGCCGCAACTGATCTTGGC SEQ ID NO: 31 = FG7 GTGACACGGCGGTTAGAACGCGGCTACAATTAATACATAACCCCATCCCCCTG TTGACAATTAATCATCGGCTCGTATAATGTGTGGAATTGTGAGCGGATAACAA TTTCACACAGGAAACAGGATCTACCATGCTGCCGGCGCCGAAAAACCTGGTTG TTTCTCGCGTTACCGAAGACTCTGCGCGTCTGTCTTGGACCGCGCCGGACGCG GCGTTCGACTCTTTCCTGATCCAGTACCAGGAATCTGAAAAAGTTGGTGAAGC GATCGTGCTGACCGTTCCGGGTTCTGAACGTTCTTACGACCTGACCGGTCTGA AACCGGGTACCGAATACACCGTTTCTATCTACGGTGTTNNNNNNNNNNNNNNN NNNNNNTCTAACCCGCTGTCTGCGATCTTCACCACCGGCGGTCACCATCACCA TCACCATGGCAGCGGTTCTAGTCTAGCGGCCGCAACTGATCTTGGC

TABLE 7 FN3 Domains, Linkers, and Albumin variant SEQ Clone ID NO: AA Sequence 3rd FN3 32 DAPSQIEVKDVTDTTALITWF domain of KPLAEIDGIELTYGIKDVPGD tenascin C RTTIDLTEDENQYSIGNLKPD (TN3 TEYEVSLISRRGDMSSNPAKE TFTT Fibcon 33 LDAPTDLQVTNVTDTSITVSW TPPSATITGYRITYTPSNGPG EPKELTVPPSSTSVTITGLTP GVEYWSLYALKDNQESPPLVG TQTT 10^(th) FN3 34 VSDVPRDLEWAATPTSLLISW domain of DAPAVTVRYYRITYGETGGNS fibronectin PVQEFTVPGSKSTATISGLKP GVDYTITVYAVTGRGDSPASS KPISINYRT Linker 35 GSGS Linker 36 GGGSGGGS Linker 37 GGGGSGGGGSGGGGSGGGGSG GGGS Linker 38 APAP Linker 39 APAPAPAPAP Linker 40 APAPAPAPAPAPAPAPAPAP Linker 41 APAPAPAPAPAPAPAPAPAP APAPAPAPAPAPAPAPAPAP Linker 42 EAAAKEAAAKEAAAKEAAAK EAAAKAAA Albumin 43 DAHKSEVAHRFKDLGEENFKA variant LVLIAFAQYLQQSPFEDHVKL VNEVTEFAKTCVADESAENCD KSLHTLFGDKLCTVATLRETY QGEMADCCAKQEPERNECFLH KKDDNPNLPRLVRPEVDVMCT AFHDNEETFLKYLYEIARRHP YFYAPELLFFAKRYKAAFTEC CQAADKAACLLPKLDELRDEG KASSAKQRLKCASLQKFGERA FKAWAVARLSQRFPKAEFAEV SKLVTDLTKVHTECCHGDLLE CADDRADLAKYICENQDSISS KLKECCEKPLLEKSHCIAEVE NDEMPADLPSLAADFVESKDV CKKYAEAKDVFLGMFLYEYAR RHPDYSVVLLLRLAKTYETTL EKCCAAADPHECYAKVFDEFK PLVEEPQNLIKQNCELFEQLG EYKFQNALLVRYTKKVPQVST PTLVEVSRNLGKVGSKCCKHP EAKRMPCAEDYLSVVLNQLCV LHEKTPVSDRVTKCCTESLVN RRPCFSALEVDETYVPKEFNA ETFTFHADICTLSEKERQIKK QTALVELVKHKPKATKEQLKA VMDDFAAFVEKCCKADDKETC FAEEGKKLVAASQAALGL

SEQ ID NO: 44 = humanCD137 MGNSCYNIVATLLLVLNFERTRSLQDPCSNCPAGTFCDNNRNQICSPCPPNSFSSA GGQRTCDICRQCKGVFRTRKECSSTSNAECDCTPGFHCLGAGCSMCEQDCKQGQEL TKKGCKDCCFGTFNDQKRGICRPWTNCSLDGKSVLVNGTKERDVVCGPSPADLSPG ASSVTPPAPAREPGHSPQIISFFLALTSTALLFLLFFLTLRFSVVKRGRKKLLYIF KQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL CD137 binding fibronectin type III domains ISOP120AR5P1D2 SEQ ID NO: 45 LPAPKNLVVSRVTEDSARLSWDFAYFKFDSFLIQYQESEKVGEAIVLTVPGSERSY DLTGLKPGTEYTVSIYGVGRFYTVYYSNPLSAIFTT ISOP120AR5P1C3 SEQ ID NO: 46 LPAPKNLVVSRVTEDSARLSWSPVDADFTFDSFLIQYQESEKVGEAIVLTVPGSER SYDLTGLKPGTEYTVSIYGVGRHYTVYDSNPLSAIFTT ISOP120AR5P1H3 SEQ ID NO: 47 LPAPKNLVVSRVTEDSARLSWKWISHEPLEFDSFLIQYQESEKVGEAIVLTVPGSER SYDLTGLKPGTEYTVSIYGVGRHYTVYDSNPLSAIFTT ISOP120AR5P1C4 SEQ ID NO: 48 LPAPKNLVVSRVTEDSARLSWAFQWHIFDSFLIQYQESEKVGEAIVLTVPGSERSY DLTGLKPGTEYTVSIYGVGQPYTVYDSNPLSAIFTT ISOP120AR5P1B5 SEQ ID NO: 49 LPAPKNLVVSRVTEDYARLSWKYGEHIIWFDSFLIQYQESEKVGEAIVLTVPGSER SYDLTGLKPGTEYTVSIYGVKGQHDHDSNPLSAIFTT ISOP120AR5P1C5 SEQ ID NO: 50 LPAPKNLVVSRVTEDSARLSWTLPNIHFDSFLIQYQESEKVGEAIVLTVPGSERSYD LTGLKPGTEYTVSIYGVGRHYTVYDSNPLSAIFTT ISOP120AR5P1G7 SEQ ID NO: 51 LPAPKNLVVSRVTEDSARLSWSQHYLSPIPFDSFLIQYQESEKVGEAIVLTVPGSER SYDLTGLKPGTEYTVSIYGVGRHYTVYDSNPLSAIFTT ISOP120AR5P1G8 SEQ ID NO: 52 LPAPKNLVVSRVTEDSARLSWHATFGDPFDSFLIQYQESEKVGEAIVLTVPGSERS YDLTGLKPGTEYTVSIYGVGRHYTVYDSNPLSAIFTT ISOP120AR5P1D9 SEQ ID NO: 53 LPAPKNLVVSRVTEDSARLSWNTDWVHTFDSFLIQYQESEKVGEAIVLTVPGSERS YDLTGLKPGTEYTVSIYGVGRHYTVYDSNPLSAIFTT ISOP120AR5P1F11 SEQ ID NO: 54 LPAPKNLVVSRVTEDSARLSWTNEQITKYGFDSFLIQYQESEKVGEAIVLTVPGSE RSYDLTGLKPATEYTVSIYGVGRHYTVYDSNPLSAIFTT ISOP120AR5P1B12 SEQ ID NO: 55 LPAPKNLVVSRVTEDSARLSWDGDKWANFKFDSFLIQYQESEKVGEAIVLTVPGS ERSYDLTGLKPGTEYTVSIYGVGLHYIVYDSNPLSAIFTT ISOP120BR5P1C1 SEQ ID NO: 56 LPAPKNLVVSRVTEDSARLSWVREDAYAFDSFLIQYQESEKVGEAIVLTVPGSERS YDLTGLKPGTEYTVSIYGVSSLHWVVHDSNPLSAIFTT ISOP120BR5P1F1 SEQ ID NO: 57 LPAPKNLVVSRVTEDSARLSWTFHPTFEGFDSFLIQYQESEKVGEAIVLTVPGSERS YDLTGLKPGTEYTVSIYGVKWTVLRPWLSNPLSAIFTT ISOP120BR5P1D2 SEQ ID NO: 58 LPAPKNLVVSRVTEDSARLSWIRKHNHVKWFDSFLIQYQESEKVGEAIVLTVPGSE RSYDLTGLKPGTEYTVSIYGVGFLIDTDDSNPLSAIFTT ISOP120BR5P1E2 SEQ ID NO: 59 LPAPKNLVVSRVTEDSARLSWAQELDHFDSFLIQYQESEKVGEAIVLTVPGSERSY DLTGLKPGTEYTVSIYGVYWTWWVRWNSNPLSAIFTT ISOP120BR5P1F2 SEQ ID NO: 60 LPAPKNLVVSRVTEDSARLSWTFHPTFEGFDSFLIQYQESEKVGEAIVLTVPGSERS YDLTGLKPGTEYTVSIYGVKWYAGIGYPVSNPLSAIFTT ISOP120BR5P1D3 SEQ ID NO: 61 LPAPKNLVVSRVTEDSARLSWSEHPTPFATFDSFLIQYQESEKVGEAIVLTVPGSER SYDLTGLKPGTEYTVSIYGVWWVENHFPVSNPLSAIFTT ISOP120BR5P1H3 SEQ ID NO: 62 LPAPKNLVVSRVTEDSARLSWEESRQFFDSFLIQYQESEKVGEAIVLTVPGSERSY DLTGLKPGTEYTVSIYGVVHRAWLRWNGSNPLSAIFTT ISOP120BR5P1E4 SEQ ID NO: 63 LPAPKNLVVSRVTEDSARLSWDDQFEDWFDSFLIQYQESEQVGEAIVLTVPGSERS YDLTGLKPGTEYTVSIYGVHTRDWTAWNASNPLSAIFTT ISOP120BR5P1G4 SEQ ID NO: 64 LPAPKNLVVSRVTEDSARLSWAGHYRKIRNFDSFLIQYQESEKVGEAIVLTVPGSE RSYDLTGLKPGTEYTVSIYGVKFPYYYATADSNPLSAIFTT ISOP120BR5P1A5 SEQ ID NO: 65 LPAPKNLVVSRVTEDSARLSWAGHYRKIRNFDSFLIQYQESEKVGEAIVLTVPGSK RSYDLTGLKPGTEYTVSIYGVKFPYYYATADSNPLSAIFTT ISOP120BR5P1E6 SEQ ID NO: 66 LPAPKNLVVSRVTEDSARLSWLEGANAEFDSFLIQYQESEKVGEAIVLTVPGSERS YDLTGLKPGTEYTVSIYGVHWVGPWYPVSNPLSAIFTT ISOP120BR5P1B7 SEQ ID NO: 67 LPAPKNLVVSRVTEDSARLSWGAKTRQFDSFLIQYQESEKVGEAIVLTVPGSERSY DLTGLKPGTEYTVSIYGVWWVENHFPVSNPLSAIFTT ISOP120BR5P1C7 SEQ ID NO: 68 LPAPKNLVVSRVTEDSARLSWNVTQKEFDSFLIQYQESEKVGEAIVLTVPGSERSY DLTGLKPGTEYTVSIYGVGNRYYTVYDSNPLSAIFTT ISOP120BR5P1D8 SEQ ID NO: 69 LPAPKNLVVSRVTEDSARLSWKNHTQEWEFDSFLIQYQESEKVGEAIVLTVPGSER SYDLTGLKPGTEYTVSIYGVPIAWLAWTSTSNPLSAIFTT ISOP120BR5P1E8 SEQ ID NO: 70 LPAPKNLVVSRVTEDSARLSWNGGEYWVPRFDSFLIQYQESEKVGEAIVLTVPGS ERSYDLTGLKPGTEYTVSIYGVVWLQWISWTDSNPLSAIFTT ISOP120BR5P1B9 SEQ ID NO: 71 LPAPKNLVVSRVTEDSARLSWAVEFNPTKFDSFLIQYQESEKVGEAIVLTVPGSER SYDLTGLKPGTEYTVSIYGVWWFEQWYPVSNPLSAIFTT ISOP120BR5P1C9 SEQ ID NO: 72 LPAPKNLVVSRVTEDSARLSWAWNRHDFDSFLIQYQESEKVGEAIVLTVPGSERS YDLTGLKPGTEYTVSIYGVHWTVLRPFIDSNPLSAIFTT ISOP120BR5P1D9 SEQ ID NO: 73 LPAPKNLVVSRVTEDSARLSWTINSHIFDSFLIQYQESEKVGEAIVLTVPGSERSYD LTGLKPGTEYTVSIYGVWGTKYWQAQSNPLSAIFTT ISOP120BR5P1A10 SEQ ID NO: 74 LPAPKNLVVSRVTEDSARLSWTEEDITHLRFDSFLIQYQESEKVGEAIVLTVPGSER SYDLTGLKPGTEYTVSIYGVYWTWWVRWNSNPLSAIFTT ISOP120BR5P1G11 SEQ ID NO: 75 LPAPKNLVVSRVTEDSARLSWTKRHFYTFDSFLIQYQESEKVGEAIVLTVPGSERS YYLTGLKPGTENTVSIYGVHGNHPYTDAPANPLSAIFTT ISOP120GR5P1E1 SEQ ID NO: 76 LPAPKNLVVSRVTEDSARLSWTAPDAAFDSFIIQYAEDSSWGEAINLHVPGSERSY DLTGLKPGTEYHVHIYGVKGGEASNPLWAWFTT ISOP120GR5P1G3 SEQ ID NO: 77 LPAPKNLVVSRVTEDSARLSWTAPDAAFDSFYIRYWEFCHSGEAIELSVPGSERSY DLTGLKPGTEYFVRIVGVKGGRVSLPLGAKFTT ISOP120GR5P1F5 SEQ ID NO: 78 LPAPKNLVVSRVTEDSARLSWTAPDAAFDSFEIDYWEVESEGEAIVLFVPGSERSY DLTGLKPGTEYHVHIVGVKGGTPSYPLWADFTT ISOP120GR5P1H6 SEQ ID NO: 79 LPAPKNLVVSRVTEDSARLSWTNEQITKYGFDSFLIQYQESEKVGEAIVLTVPGSE RSYDLTGLKPGTEYTVSIYGVGRHYTVYDSNPLSAIFTT ISOP120GR5P1E7 SEQ ID NO: 80 LPAPKNLVVSRVTEDSARLSWTAPDAAFDSFEIDYWEVESEGEAIILFVPGSERSY DLTGLKPGTEYHVHIVGVKGGTPSYPLWADFTT ISOP120GR5P1A10 SEQ ID NO: 81 LPAPKNLVVSRVTEDSARLSWTAPDAAFDSFEIPYIEVETIGEAIWLHVPGSERSYD LTGLKPGTEYSVGINGVKGGHTSNPLSARFTT ISOP120GR5P1C10 SEQ ID NO: 82 LPAPKNLVVSRVTEDSARLSWTAPDGAFDSFEIPYIEVETIGEAIWLHVPGSERSYD LTGLKPGTEYSVGINGVKGGHTSNPLSARFTT ISOP120GR5P1A11 SEQ ID NO: 83 LPAPKNLVVSRVTEDSARLSWTAPDAAFDSFGIPYWEWTTEGEAIQLIVPGSERSY DLTGLKPATEYHVHIVGVKGGSFSEPLPADFTT ISOP120GR5P1B11 SEQ ID NO: 84 LPAPKNLVVSRVTEDSARLSWTAPDAAFDSFNIKYWEANLYGEAIVLTVPGSERS YGLTGLKPGTEYRVHIRGVKGGINSFPLVAVFTT ISOP120GR5P1H11 SEQ ID NO: 85 LPAPKNLVVSRVTEDSARLSWTAPDAAFDSFYIAYWEYWGNGEAIGLIVPGSERS YDLTGLKPGTEYHVHIVGVKGGAGSVPLWANFTT ISOP120HR5P1E2 SEQ ID NO: 86 LPAPKNLVVSHVTEDSARLSWTAPDAAFDSFEIYYLEGGRGEAIVLTVPGSERSYD LTVLKPGTEYLGTIYGVKCGWASNPLSAIFTT ISOP120HR5P1A3 SEQ ID NO: 87 LPAPKNLVVSRVTEDSARLSWTAPDAAFDSFEIYYAEFGYYGEAIVLTVPGSERSY DLTGLKPGTEYTVTIYGVKGGWYSTPLSAIFTT ISOP120HR5P1B4 SEQ ID NO: 88 LPAPKNLVVSRVTEDSARLSWTAPDAAFDSFSIYYGEYYNLGEAIVLTVPGSERSY DLTGLKPGTEYVVTIYGVKGGGYSNPLSAIFTT ISOP120HR5P1G4 SEQ ID NO: 89 LPAPKNLVVSRVTEDSARLSWTAPDAAFDSFEIYYREYWYSGEAIVLTVPGSERSY DLTGLKPGTEYLVTIYGVKGGWYSDPLSAIFTT ISOP120HR5P1H4 SEQ ID NO: 90 LPAPKNLVVSRVTEDSARLSWTAPDAAFDSFDILYLEPYQEGEAIVLTVPGSERSY DLTGLKPGTEYLVTIYGVKGGYYSLPLSAIFTT ISOP120HR5P1B5 SEQ ID NO: 91 LPAPKNLVVSRVTEDSARLSWTAPDAAFDSFIIRYIEEGYYGEAIVLTVPGSERSYD LTGLKPGTEYHVGIEGVKGGYYSYPLSAIFTT ISOP120HR5P1A6 SEQ ID NO: 92 LPAPKNLVVSRVTEDSARLSWTAPDGAFDSFEIYYLEGGRGEAIVLTVPGSERSYD LTGLKPGTEYLVTIYGIKCGWASNPLSAIFTT ISOP120HR5P1G6 SEQ ID NO: 93 LPAPKNLVVSRVTEDSARLSWTAPDAAFDSFEIYYFELRLGGEAIVLTVPGSERSY DLTGLKPGTEYLVTIYGVKGGLDSQPLSAIFTT ISOP120HR5P1A7 SEQ ID NO: 94 LPAPKNLVVSRVTEDSARLSWTAPDAAFDSFEIYYAEPRYYGEAIVLTVPGSERSY DLTGLKPGTEYTVTIYGVKGGYYSSPLSAIFTT ISOP120HR5P1D7 SEQ ID NO: 95 LPAPKNLVVSRVTEDSARLSWTAPDAAFDSFDIYYLESWTRGEAIVLTVPGSERSY DLTGLKPGTEYLVTIYGVKGGSYSRPLSAIFTT ISOP120HR5P1E7 SEQ ID NO: 96 LPAPKNLVVSRVTEDSARLSWTAPDAAFDSFQIYYLEQLGYGEAIVLTVPGSERSY DLTGLKPGTEYLVTIYGVKVCEQSYPLSAIFTT ISOP120HR5P1H7 SEQ ID NO: 97 LPAPKNLVVSRVTEDSARLSWTAPDAAFDSFDIYYGEPGNLGEAIVLTVPGSERSY DLTGLKPGTEYLVTIYGVKGGDYSSPLSAIFTT ISOP120HR5P1H8 SEQ ID NO: 98 LPAPKNLVVSRVTEDSARLSWTAPDAAFDSFDIYYYELRLGGEAIVLTVPGSERSY DLTGLKPGTEYLVTIYGVKGGYYSGPLSAIFTT ISOP120HR5P1D9 SEQ ID NO: 99 LPAPKNLVVSRVTEDSARLSWTAPDAAFDSFDIYYRELDFQGEAIVLTVPGSERSY DLTGLKPGTEYLVIIYGVKGGSYSYTLSAIFTT ISOP120HR5P1F9 SEQ ID NO: 100 LPAPKNLVVSRVTEDSARLSWTAPDAAFDSFYIYYREHWTIGEAIVLTVPGSERSY DLTGLKPGTEYLVTIYGVKGGAYSNPLSAIFTT ISOP120ER5P1B4 SEQ ID NO: 101 LPAPKNLVVSRVTEDSARLSWTAPDAAFDSFSILYGEPPALGEAIVLTVPGSERSY DLTGLKPGTEYWVTIYGVKGGVFSHPLSAIFTT ISOP120ER5P1F4 SEQ ID NO: 102 LPAPKNLVVSRVTEDSARLSWTAPDAAFDSFVIRYIEDTVMGEAIVLTVPGSERSY DLTGLKPGTEYHVSIEGVKGGPSSLPLSAIFTT ISOP120ER5P1H4 SEQ ID NO: 103 LPAPKNLVVSRVTEDSARLSWTAPDAAFDSFNIMYLEDVQCGEAIVLTVPGSERS YDLTGLKPGTEYHVGINGVKGGLRSFPLSAIFTT ISOP120ER5P1E5 SEQ ID NO: 104 LPAPKNLVVSRVTEDSARLSWTAPDAAFDSFRISYLEDVYYGEAIVLTVPGSERSY DLTGLKPGTEYHVGIHGVKGGIDSFPLSAIFTT ISOP120ER5P1B6 SEQ ID NO: 105 LPAPKNLVVSRVTEDSARLSWTAPDAAFDSFDIYYGEHWKLGEAIVLTVQGSERS YDLTGLKPGTEYLVTIYGVKGGQWSFPLSAIFTT ISOP120ER5P1C6 SEQ ID NO: 106 LPAPKNLVVSRVTEDSARLSWTAPDAAFDSFSIYYGEWHALGEAIVLTVPGSERSY DLTGLKPGTEYVVTIYGVKGGTYSLPLSAISTT ISOP120ER5P1H6 SEQ ID NO: 107 LPAPKNLVVSRVTEDSARLSWTAPDAAFDSFSIYYGEWHALGEAIVLTVPGSERSY DLTGLKPGTEYVVTIYGVKGGTYSLPLSAIFTT ISOP120ER5P1A7 SEQ ID NO: 108 LPAPKNLVVSRVTEDSARLSWTAPDAAFDSFNIGYYERIIPGEAIVLTVPGSERSYD LTGLKPGTEYSVLICGVKGGKGSIPLSAIFTT ISOP120ER5P1A8 SEQ ID NO: 109 LPAPKNLVVSRVTEDSARLSWTAPDAAFDSFDIYYFEHPVGGEAIVLTVPGSERSY DLTGLKPGTEYLVTIYGVKGGYLSMPLSAIFTT ISOP120ER5P1E10 SEQ ID NO: 110 LPAPKNLVVSRVTEDSARLSWTAPDAAFDSFEIYYMEDFHSGEAIVLTVPGSERSY DLTGLKPGTEYWVTIYGVEGGTGSLPLSAIFTT ISOP120ER5P1A11 SEQ ID NO: 111 LPAPKNLVVSRVTEDSARLSWTAPDAAFDSFEIYYKELRAEGEAIVLTVPGSERSY DLTGLKPGTEYLVTIYGVKGGSVSIPLSAIFTT ISOP120ER5P1B12 SEQ ID NO: 112 LPAPKNLVVSRVTEDSARLSWTAPDAAFDSFAIYYIEWTAYGEAIVLTVPGSERSY DLTGLKPGTEYVVRISGVKCGIVSFPLSAIFTT ISOP120FR5P1F1 SEQ ID NO: 113 LPAPKNLVVSRVTEDSARLSWTAPDAAFDSFTIYYFENENGGEAIVLTVPGSERSY DLTGLKPGTEYLVTIYGVKGCDWSDPLSAIFTT ISOP120FR5P1C2 SEQ ID NO: 114 LPAPKNLVVSRVTEDSARLSWTAPDAAFDSFDINYFEQPKGGEAIVLTVPGSERSY DLTGLKPGTEYLVTIYGVKGGPYSPPLSAIFTT ISOP120FR5P1H5 SEQ ID NO: 115 LPAPKNLVVSRVTEDSARLSWTAPDAAFDSLQIYYFEWVVGGEAIVLTVPGSERS YDLTGLKLGTEYLVTIYGVKGGNFSDPLSAIFTT ISOP120FR5P1A6 SEQ ID NO: 116 LPAPKNLVVSRVTEDSARLSWTAPDAAFDSFIIRYLEDISYGEAIVLTVPGSERSYD LTGLKPGTEYHVGIEGVKGGNVSFPLSAIFTT ISOP120FR5P1H6 SEQ ID NO: 117 LPAPKNLVVSRVTEDSARLSWTAPDAAFDSFGIPYLEDIEVGEAIVLTVPGSERSYD LAGLKPGTEYHVGIYGVKGGEQSFPLSAIFTT ISOP120FR5P1D7 SEQ ID NO: 118 LPAPKNLVVSRVTEDSARLSWTAPDAAFDSFIIRYLEDISYGEAIVLTVPGSERSYD LTGLKPGTEYHVGIEGVKGGNVSWPLSAIFTT ISOP120FR5P1F8 SEQ ID NO: 119 LPAPKNLVVSRVTEDSARLSWTAPDAAFDSFIIRYLEDISYGEAIVLTVPGSERSYD LTGLKPGTEYHVGIEGVKGGNVSWPLSAIFTT ISOP120FR5P1E9 SEQ ID NO: 120 LPAPKNLVVSRVTEDSARLSWTAPDAAFDSFIIYYPEYISNGEAIVLTVPGSERSYD LTGLKPGTEYHVTIGVKGGHSWPLSAIFTT ISOP120FR5P1E10 SEQ ID NO: 121 LPAPKNLVVSRVTEDSARLSWTAPDAAFDSFLIHYTEQPSKGEAIVLTVPGSERSY DLTGLKPGTEYQVPIGVKGGTQSCPLSAIFTT ISOP120FR5P1A11 SEQ ID NO: 122 LPAPKNLVVSRVTEDSARLSWTAPDAAFDSFTIYYFENENGGEAIVLTVPGSERSY DLTGLKPGTEYLVTIYGVKGGHWSRPLSAIFTT ISOP193AR9P1A11 SEQ ID NO: 123 LPAPKNLVVSRVTEDSARLSWALSSVHAYFDSFLIQYQESEKVGEAIVLTVPGSER SYDLTGLKPGTEYTVSIYGVQYVDGFFKSNPLSAIFTT ISOP193AR9P1A6 SEQ ID NO: 124 LPAPKNLVVSRVTEDSARLSWKFGEVAFDSFLIQYQESEKVGEAIVLTVPGSERSY DLTGLKPGTEYTVSIYGVGRHYTVYDSNPLSAIFTT ISOP193AR9P1B10 SEQ ID NO: 125 LPAPKNLVVSRVTEDSARLSWAFQWHIFDSFLIQYQESEKVGEAIVLTVPGSERSY DLTGLKPGTEYTVSIYGVGRHYTVYDSNPLSAIFTT ISOP193AR9P1B12 SEQ ID NO: 126 LPAPKNLVVSRVTEDSARLSWTNEQITKYGFDSFLIQYQESEKVGEAIVLTVPGSE RSYDLTGLKPGTEYTVSIYGVGAPYTVYDSNPLSAIFTT ISOP193AR9P1B4 SEQ ID NO: 127 LPAPKNLVVSRVTEDSARLSWRDLQYHTFDSFLIQYQESEKVGEAIVLTVPGSERS YDLTGLKPGTEYTVSIYGVGRHYTVYDSNPLSAIFTT ISOP193AR9P1C10 SEQ ID NO: 128 LPAPKNLVVSRVTEDSARLSWPNHISIFDSFLIQYQESEKVGEAIVLTVPGSERSYD LTGLKPGTEYTVSIYGVGRFYTVFDSNPLSAIFTT ISOP193AR9P1E6 SEQ ID NO: 129 LPAPKNLVVSRVTEDSARLSWKFHSPTFDSFLIQYQESEKVGEAIVLTVPGSERSY DLTGLKPGTEYTVSIYGVGRHYTVYDSNPLSAIVTT ISOP193AR9P1F4 SEQ ID NO: 130 LPAPKNLVVSRVTEDSARLSWLEQEQFVNHFDSFLIQYQESEKVGEAIVLTVPGSE RSYDLTGLKPGTEYTVSIYGVQYVDGFFKSNPLSAIFTT ISOP193AR9P1F9 SEQ ID NO: 131 LPAPKNLVVSRVTEDSARLSWPLFASDLNIFDSFLIQYQESEKVGEAIVLTVPGSER SYDLTGLKPGTEYTVSIYGVGRHYTVYDSNPLSAIFTT ISOP193AR9P1G11 SEQ ID NO: 132 LPAPKNLVVSRVTEDSARLSWTNEQITKYGFDSFLIQYQESEKVGEAIVLTVPGSE RSYDLTGLKPGTEYTVSIYGVGRHYTVYDSNPLSAIFTT ISOP193AR9P1G5 SEQ ID NO: 133 LPAPKNLVVSRVTEDSARLSWRISDRLPLFDSFLIQYQESEKVGEAIVLTVPGSERS YDLTGLKPGTEYTVSIYGVGRHYTVYDSNPLSAIFTT ISOP193AR9P1G8 SEQ ID NO: 134 LPAPKNLVVSRVTEDSARLSWHATFGDPFDSFLIQYQESEKVGEAIVLTVPGSERS YDLTGLKPGTEYTVSIYGVGRHYTVYDSNPLSAIFTT ISOP193AR9P1H8 SEQ ID NO: 135 LPAPKNLVVSRVTEDSARLSWTNEQITKYGFDSFLIQYQESEKVGEAIVLTVPGSE RSYDLTGLKPGTEYTVSIYGVGRFYTVFDSNPLSAIFTT ISOP193BR9P1B10 SEQ ID NO: 136 LPAPKNLVVSRVTEDSARLSWAWNRHDFDSFLIQYQESEKVGEAIVLTVPGSERS YDLTGLKPGTEYTVSIYGVHWTVLRPFIDSNPLSAIFTT ISOP193BR9P1B12 SEQ ID NO: 137 LPAPKNLVVSRVTEDSARLSWPDESRPVRFDSFLIQYQESEKVGEAIVLTVPGSERS YDLTGLKPGTEYTVSIYGVLRPWIYATNDSNPLSAIFTT ISOP193BR9P1E6 SEQ ID NO: 138 LPAPKNLVVSRVTEDSARLSWGAITALFDSFLIQYQESEKVGEAIVLTVPGSERSY DLTGLKPGTEYTVSIYGVKFPYYYATADSNPLSAIFTT ISOP193BR9P1G11 SEQ ID NO: 139 LPAPKNLVVSRVTEDSARLSWAGHYRKIRNFDSFLIQYQESEKVGEAIVLTVPGSE RSYDLTGLKPGTEYTVSIYGVKFPYYYATADSNPLSAIFTT ISOP193BR9P1G2 SEQ ID NO: 140 LPAPKNLVVSRVTEDSARLSWAGHYRKIRNFDSFLIQYQESEKVGEAIVLTVPGSE RSYDLTGLKPGTEYTVSIYGVAEHWYYATQDSNPLSAIFTT ISOP193BR9P1G3 SEQ ID NO: 141 LPAPKNLVVSRVTEDSARLSWAQSNQQFDSFLIQYQESEKVGEAIVLTVPGSERSY DLTGLKPGTEYTVSIYGVVWQNWVAYNSNPLSAIFTT ISOP193BR9P1G6 SEQ ID NO: 142 LPAPKNLVVSRVTEDSARLSWDDQFEDWFDSFLIQYQESEQVGEAIVLTVPGSERS YDLTGLKPGTEYTVSIYGVHTRDWTAWNASNPLSAIFTT ISOP193BR9P1G9 SEQ ID NO: 143 LPAPKNLVVSRVTEDSARLSWKQVTVAPEFDSFLIQYQESEKVGEAIVLTVPGSER SYDLTGLKPGTEYTVSIYGVKFPYYYATADSNPLSAIFTT ISOP193BR9P1H2 SEQ ID NO: 144 LPAPKNLVVSRVTEDSARLSWPDESRPVRFDSFLIQYQESEKVGEAIVLTVPGSERS YDLTGLKPGTEYTVSIYGVHTRDWTAWNASNPLSAIFTT ISOP193BR9P1H3 SEQ ID NO: 145 LPAPKNLVVSRVTEDSARLSWNRLDSEWVAFDSFLIQYQESEKVGEAIVLTVPGSE RSYDLTGLKPGTEYTVSIYGVVFRPWLAYNSNPLSAIFTT ISOP193BR9P1H6 SEQ ID NO: 146 LPAPKNLVVSRVTEDSARLSWPDESRPVRFDSFLIQYQESEKVGEAIVLTVPGSERS YDLTGLKPGTEYTVSIYGVVGQWKYATADSNPLSAIFTT ISOP193ER9P1A10 SEQ ID NO: 147 LPAPKNLVVSRVTEDSARLSWTAPDAAFDSFDIYYFEHPVGGEAIVLTVPGSERSY DLTGLKPGTEYLVTIYGVKGGHFSGPLSAIFTT ISOP193ER9P1A11 SEQ ID NO: 148 LPAPKNLVVSRVTEDSARLSWTAPDAAFDSFAIQYQEYVAHGEAIVLTVPGSERS YDLTGLKPGTEYHVRISGVKGGGVSWPLSAIFTT  ISOP193ER9P1A3 SEQ ID NO: 149 LPAPKNLVVSRVTEDSARLSWTTPDAAFDSFDIYYFEHPVGGEAIVLTVPGSERSY DLTGLKPGTEYLVTIYGVKGGYLSKPLSAIFTT ISOP193ER9P1A4 SEQ ID NO: 150 LPAPKNLIVSRVTEDSARLSWTAPDAAFDSFEIYYKELRAEGEAIVLTVPGSERSYD LTGLKPGTEYLVTIYGVKGGSVSIPLSAIFTT ISOP193ER9P1A8 SEQ ID NO: 151 LPAPKNLVVSRVTEDSARLSWTAPDAAFDSLDIYYFEHPVGGEAIVLTVPGSERSY DLTGLKPGTEYLVTIYGVKGGYLSMPLSAIFTT ISOP193ER9P1B4 SEQ ID NO: 152 LPAPKNLVVSHVTEDSARLSWTAPDAAFDSFDIYYFEHPVGGEAIVLTVPGSERSY DLTGLKPGTEYLVTIYGVKGGYLSMPLSAIFTT ISOP193ER9P1B5 SEQ ID NO: 153 LPAPKNLVVSRVTEDSARLSWTAPDAAFDSFAIQYQEYVAHGEAIVLTVPGSERS YDLTGLKPGTEYHVRISGVKGGGVSWPLSAIVTT ISOP193ER9P1C10 SEQ ID NO: 154 LLAPKNLVVSRVTEDSARLSWIAPDAAFDSFEIYYKELRAEGEAIVLTVPGSERSY DLTGLKPGTEYLVTIYGVKGGSVSIPLSAIFTT ISOP193ER9P1C4 SEQ ID NO: 155 LPAPKNLVVSRVTEDSARLSWTAPDAAFDSFDIYYFEHPVGGEAIVLTVPGSERSY DLTGLKPGTEYLVTIYGVKGGIWSVPLSAIFTT ISOP193ER9P1C8 SEQ ID NO: 156 LPAPKNLVVSRVTEDSARLSWTAPDAAFDSFDIYYFEHPVGGEAIVLTVPGSERSY DLTGLKPGTEYLVTIYGVKGGTYSLPLSAIFTT ISOP193ER9P1C9 SEQ ID NO: 157 LPAPKNLVVSRVTEDSARLSWTAPDAAFDSFDIYYFEHPVGGEAIVLTVPGSERSY DLTGLKPGTEYLVTIYGVKGGELSKPLSAISTT ISOP193ER9P1D4 SEQ ID NO: 158 LPAPKNLVVSRVTEDSARLSWTAPDAAFDSFDIYYFEHPVGGEAIVLTVPGSERSY DLTGLKPGTEYLVTIYGVKGGTYSPPLSAIFTT ISOP193ER9P1D7 SEQ ID NO: 159 LPAPKNLVVSRVTEDSARLSWTAPDAAFDSFDIYYGEHWKLGEAIVLTVPGSERS YDLTGLKPGTEYLVTIYGVKGGMSSNPLSAIFTT ISOP193ER9P1E1 SEQ ID NO: 160 LPAPKNLVVSRVTEDSARLSWTAPDAAFDSFDIYYFEHPVGGEAIVLTVPGSERSY DLTGLKPGTEYLVTIYGVKGGSVSIPLSAIFTT ISOP193ER9P1E2 SEQ ID NO: 161 LPAPKNLVVSRVTEDSARLSWTAPDAAFDSFDIYYFEHPVGGEAIVLTVPGSERSY DLTGLKPGTEYLVTIYGVKGGFWSQPLSAIFTT ISOP193ER9P1E4 SEQ ID NO: 162 LPAPKNLVVSRVTEDSARLSWTAPDAAFDSFAIIYQEYVKSGEAIVLTVPGSERSY DLTGLKPGTEYHVRIGGVKGGLLSLPLSAIFTT ISOP193ER9P1E8 SEQ ID NO: 163 LPAPKNLVVSRVTEDSARLSWTAPDAAFDSFDIYYFEHPVGGEAIVLTVPGSERSY DLTGLKPGTEYLVTIYGVKGGELSKPLSAIFTT ISOP193ER9P1F11 SEQ ID NO: 164 LPAPKNLVVSRVTEDSARLSWTAPDAAFDSFAIQYQEYVAHGEAIVLTVPGSERS YDLTGLKPGTEYHVRISGVKGGGVSWPLSAISTT ISOP193ER9P1F7 SEQ ID NO: 165 LPAPKNLVVSRVTEDSAHLSWTAPDAAFDSFDIYYFEHPVGGEAIVLTVPGSERSY DLTGLKPGTEYLVTIYGVKGGYLSMPLSAIFTT ISOP193ER9P1F9 SEQ ID NO: 166 LPAPKNLVVSRVTEDSARLSWTAPDAAFDSFHIYYGEHYNLGEAIVLTVPGSERSY DLTGLKPGTEYLVTIYGVKGGFWSTPLSAIFTT ISOP193ER9P1G11 SEQ ID NO: 167 LPAPKNLVVSRVTEDSARLSWTAPDAAFDSFDIYYFEHPVGGEAIVLTVPGSERSY DLTGLKPGTEYLVTIYGVKGGYLSMPLSAIFTT ISOP193ER9P1G2 SEQ ID NO: 168 LPAPKNLVVSRVTEDSARLSWTAPDAAFDSFDIYYFEQPVGGEAIVLTVPGSERSY DLTGLKPGTEYLVTIYGVKGGYLSMPLSAIFTT ISOP193ER9P1G4 SEQ ID NO: 169 LPAPKNLVVSRVTEDSARLSWTAPDAAFDSFDIYYFEHPVGGEAIVLTVPGSERSY DLTGLKPGTEYLVTIYGVKGGNFSFPLSAIFTT ISOP193ER9P1G5 SEQ ID NO: 170 LPAPKNLVVSRVTEDSARLSWTAPDAAFDSFDIYYFEHPVGGEAIVLTVPGSERSY DLTGLKPGTEYLVTIYGVKGGYLSMPLSAIFTT ISOP193ER9P1G9 SEQ ID NO: 171 LPAPKNLVVSRVTEDSARLSWTAPDAAFDSFDIYYFEHPVGGEAIVLTVPGSERSY DLTGLKPGTEYLVTIYGVKGGNGSSPLSAIFTT ISOP193ER9P1H11 SEQ ID NO: 172 LPAPKNLVVSRVTEDSARLSWTAPDAAFDSFDIYYFEHPVGGEAIVLTVPGSERSY DLTGLKPGTEYLVAIYGVKGGVFSHPLSAIFTT ISOP193ER9P1H2 SEQ ID NO: 173 LPAPKNLVVSRVTEDSARLSWTAPDAAFDSFRISYLEDVYYGEAIVLTVPGSERSY DLTGLKPGTEYHVGIHGVKGGIDSFPLSAIFTT ISOP193ER9P1H3 SEQ ID NO: 174 LPAPKNLVVSRVTEDSARLSWTAPDAAFDSFEIYYLEVRNRGEAIVLTVPGSERSY DLTGLKPGTEYHVGIAGVKGGFHSFPLSAIFTT ISOP193FR9P1A11 SEQ ID NO: 175 LPAPKNLVVSRVTEDSARLSWTAPDAAFDSFAIQYWEGWEWGEAIVLTVPGSERS YDLTGLKPGTEYLVTIYGVKGGHWSRPLSAIFTT ISOP193FR9P1A5 SEQ ID NO: 176 LPAPKNLVVSRVTEDSARLSWTAPDAAFDSFEIYYIEPIAPGEAIVLTVPGSERSYD LTGLKPGTEYWVTIYGVKGCDWSDPLSAIFTT ISOP193FR9P1C1 SEQ ID NO: 177 LPAPKNLVVSRVTEDSARLSWTAPDAAFDSFEIYYFENENGGEAIVLTVPGSERSY DLTGLKPGTEYLVTIYGVKGCDWSDPLSAIFTT ISOP193FR9P1C5 SEQ ID NO: 178 LPAPKNLVVSRVTEDSARLSWTAPDAAFDSFHIYYLEQYSRGEAIVLTVPGSERSY DLTGLKPGTEYLVTIYGVKGCDWSDPLSAIFTT ISOP193FR9P1C9 SEQ ID NO: 179 LPAPKNLVVSRVTEDSARLSWTAPDAAFDSFQIYYFEWVVGGEAIVLTVPGSERS YDLTGLKPGTEYLVTIYGVKGCDWSDPLSAIFTT ISOP193FR9P1D1 SEQ ID NO: 180 LPAPKNLVVSRVTEDSARLSWTAPDAAFDSFFIQYLEDVTNGEAIVLTVPGSERSY DLTGLKPGTEYRVPIAGVKGGRDSQPLSAIFTT ISOP193FR9P1D5 SEQ ID NO: 181 LPAPKNLVVSRVTEDSARLSWTAPDAAFDSFYIRYIEDVDFGEAIVLTVPGSERSY DLTGLKPGTEYLVTIYGVKGCDWSDPLSAIFTT ISOP193FR9P1D7 SEQ ID NO: 182 LPAPKNLVVSRVTEDSARLSWTAPDAAFDSFEIYYAEYFKNGEAIVLTVPGSERSY DLTGLKPGTEYLVTIYGVKGCDWSDPLSAISTT ISOP193FR9P1E1 SEQ ID NO: 183 LPAPKNLVVSRVTEDSARLSWTAPDAAFDSFIIRYLEDISYGEAIVLTVPGSERSYD LTGLKPGTEYHVGIEGVKGGNVSWPLSAIFTT ISOP193FR9P1E10 SEQ ID NO: 184 LPAPKNLVVSRVTEDSARLSWTTPDAAFDSFHIHYLEGEWGGEAIVLTVPGSERSY DLTGLKPGTEYLVTIYGVKGCDWSDPLSAIFTT ISOP193FR9P1F8 SEQ ID NO: 185 LPAPKNLVVSRVTEDSARLSWTAPDAAFDSFDINYFENELGGEAIVLTVPGSERSY DLTGLKPGTEYLVTIYGVKGCDWSDPLSAIFTT ISOP193FR9P1G10 SEQ ID NO: 186 LPAPKNLVVSRVTEDSARLSWTAPDAAFDSFTIYYFENENGGEAIVLTVPGSERSY DLTGLKPGTEYLVTIYGVKGGHWSRPLSAIFTT ISOP193FR9P1G11 SEQ ID NO: 187 LPAPKNLVVSRVTEDSARLSWTAPDAAFDSFTIYYFENENGGEAIVLTVPGSERSY DLTGLKPGTEYLVTIYGVKGCDWSDPLSAIFTT ISOP193FR9P1G2 SEQ ID NO: 188 LPAPKNLVVSRVTEDSARLSWTAPDAAFDSFTIYYFENENGGEAIVLTVPGSERSY DLTGLKPDTEYLVTIYGVKGGHWSRPLSAIFTT ISOP193FR9P1G4 SEQ ID NO: 189 LPAPKNLVVSRVTEDSARLSWTAPDAAFDSFEIYYFENELGGEAIVLTVPGSERSY DLTGLKPGTEYLVTIYGVKGGDWSDPLSAIFTT ISOP193FR9P1G7 SEQ ID NO: 190 LPAPKNLVVSRVTEDSARLSWTAPDAAFDSFHIHYLEGEWGGEAIVLTVPGSERS YDLTGLKPGTEYLVTIYGVKGCDWSDPLSAIFTT ISOP193FR9P1G8 SEQ ID NO: 191 LPAPKNLVVSRVTEDSARLSWTAPDAAFDSFEIYYFENELGGEAIVLTVPGSERSY DLTGLKPGTEYLVTIYGVKGCDWSDPLSAIFTT ISOP193FR9P1G9 SEQ ID NO: 192 LPAPKNLFVSRVTEDSARLSWTAPDAAFDSFQIYYREQWWDGEAIVLTVPGSERS YDLTGLKPGTEYLVTIYGVKGCDWSDPLSAIFTT ISOP193FR9P1H6 SEQ ID NO: 193 LPAPKNLVVSRVTEDSARLSWTAPDAAFDSFDINYFEQPKGGEAIVLTVPGSERSY DLTGLKPGTEYLVTIYGVKGCDWSDPLSAIFTT ISOP193FR9P1H9 SEQ ID NO: 194 LPAPKNLVVSRVTEDSARLSWTAPDAAFDSFEIYYDELRNPGEAIVLTVPGSERSY DLTGLKPGTEYAVTIYGVKGGRYSPPLSAIFTT ISOP193GR9P1A7 SEQ ID NO: 195 LPAPKNLVVSRVTEDSARLSWTAPDAAFDSFAIFYHEFANPGEAIDLPVPGSERSY DLTGLKPGTEYDVRIYGVKGGTASIPLDAEFTT ISOP193GR9P1B3 SEQ ID NO: 196 LPAPKNLVVSRVTEDSARLSWTAPDAAFDSFAIGYVEWTANGEAIVLIVPGSERSY DLTGLKPGTEYVVRIRGGVKGGDSSFPLRADFTT ISOP193GR9P1E10 SEQ ID NO: 197 LPAPKNLVVSRVTEDSARLSWTAPDAAFDSFAISYTESIRQGEAIWLWVPGSERSY DLTGLKPGTEYEVTIGGVKGGIRSYPLWAWFTT ISOP193GR9P1F6 SEQ ID NO: 198 LPAPKNLVVSRVTEDSARLSWTAPDAAFDSFEIDYWEVESEGEAIVLFVPGSERSY DLTGLKPGTEYHVHIVGVKGGTPSYPLWADFTT ISOP193GR9P1F7 SEQ ID NO: 199 LPAPKNLVVSRVTEDSARLSWTAPDAAFDSFDIPYVEYYPSGEAIVLNVPGSERSY DLTGLKPGTEYGVTIWGIKGGNESVPLTARFTT ISOP193GR9P1G9 SEQ ID NO: 200 LPAPKNLVVSRVTEDSARLSWTAPDAAFDSFAIFYHEFANSGEAIDLPVPGSERSY DLTGLKPGTEYDVRIYGVKGGTASIPLDAEFTT ISOP193GR9P1H2 SEQ ID NO: 201 LPAPKNLVVSRVTEDSARLSWTAPDAAFDSFEIPYIEVETIGEAIWLHVPGSERSYD LTGLKPGTEYSVGINGVKGGHTSNPLSARFTT ISOP193HR9PlA10 SEQ ID NO: 202 LPAPKNLVVSRVTEDSARLSWTAPDAAFDSFDIYYLEPWGGGEAIVLTVPGSERSY DLTGLKPGTEYWVTIYGVKVCLGSNPLSAIFTT ISOP193HR9PlA11 SEQ ID NO: 203 LPAPKNLVVSRVTEDSARLSWTAPDAAFDSFEIYYLEGGRGEAIVLTVPGSERSYD LTGLKPGTEYLVTIYGVKCGWASNPLSAIFTT ISOP193HR9P1A5 SEQ ID NO: 204 LPAPKNLVVSRVTEDSARLSWTAPDAAFDSFDIYYYELRLGGEAIVLTVPGSERSY DLTGLKPGTEYLVTIYGVKGCGYSAPLSAIVTT ISOP193HR9P1A6 SEQ ID NO: 205 LPAPKNLVVSRVTEDSARLSWTAPDAAFDSFDIYYYELRLGGEAIVLTVPGSERSY DLTGLKPGTEYLVSIYGVKGCGYSDPLSAIFTT ISOP193HR9P1A7 SEQ ID NO: 206 LPAPKNLVVSRVTEDSARLSWTAPDAAFDSFDIYYYELRLGGEAIVLTVPGSERSY DLTGLKPGTEYLVTIYGVKVCNASTPLSAIFTT ISOP193HR9P1B11 SEQ ID NO: 207 LPAPKNLVVSRVTEDSARLSWTAPDAAFDSFEIYYFELRLGGEAIVLTVPGSERSY DLTGLKPGTEYLVTIYGVKGCGYSDPLSAIFTT ISOP193HR9P1B7 SEQ ID NO: 208 LPAPKNLVVSRVTEDSARLSWTAPDAAFDSFEIYYFELRLGGEAIVLTVPGSERSY DLTGLKPGTEYLVSIYGVKGCGYSDPLSAIFTT ISOP193HR9P1C7 SEQ ID NO: 209 LPAPKNLVVSRVTEDSARLSWTAPDAAFDSFEIYYLELDSDGEAIVLTVPGSERSY DLTGLKPGTEYIVTIYGVKVCTGSRPLSAIFTT ISOP193HR9P1C8 SEQ ID NO: 210 LPAPKNLVVSRVTEDSARLSWTAPDAAFDSFEIYYFELRLGGEAIVLTVPGSERSY DLTGLKPGTEYLVTIYGVKGGGYSTPLSAIFTT ISOP193HR9P1D11 SEQ ID NO: 211 LPAPKNLVVSRVTEDSARLSWTAPDAAFDSFDIYYYELRLGGEAIVLTVPGSERSY DLTGLKPGTEYLVTIYGVKVCEQSYPLSAIFTT ISOP193HR9P1D8 SEQ ID NO: 212 LPAPKNLVVSRVTEDSARLSWTAPDAAFDSFDIYYLESGRDGEAIVLTVPGSERSY DLTGLKPGTEYLVSIYGVKGCGYSDPLSAIFTT ISOP193HR9P1E2 SEQ ID NO: 213 LPAPKNLVVSRVTEDSARLSWTAPDAAFDSFEIYYLEWCSGGEAIVLTVPGSERSY DLTGLKPGTEYLVTIYGVKGCAASDPLSAIFTT ISOP193HR9P1E3 SEQ ID NO: 214 LPAPKNLVVSRVTEDSARLSWTAPDAAFDSFEIYYFELRLGGEAIVLTVPGSERSY DLTGLKPGTEYLVTIYGVKGGAYSNPLSAIFTT ISOP193HR9P1E6 SEQ ID NO: 215 LPAPKNLVVSRVTEDSARLSWTAPDAAFDSFEIYYAEFGYYGEAIVLTVPGSERSY DLTGLKPGTEYLVTIYGVKGCAASDPLSAIFTT ISOP193HR9P1E8 SEQ ID NO: 216 LPAPKNLVVSRVTEDSARLSWTAPDAAFDSFDIYYYELRLGGEAIVLTVPGSERSY DLTGLKPGTEYLVTIYGVKGGDYSPPLSAIFTT ISOP193HR9P1F10 SEQ ID NO: 217 LPAPKNLVVSRVTEDSARLSWTAPDAAFDSFDIYYYELRLGGEAIVLTVPGSERSY DLTGLKPGTEYLVTIYGVKGGYYSGPLSAIFTT ISOP193HR9P1F8 SEQ ID NO: 218 LPAPKNLVVSRVTEDSARLSWTAPDAAFDSFDIYYYELRLGGEAIVLTVPGSERSY DLTGLKPGTEYLVTIYGVKVCYYSTPLSAIFTT ISOP193HR9P1G10 SEQ ID NO: 219 LPAPKNLVVSRVTEDSARLSWTAPDAAFDSFEIYYFELRLGGEAIVLTVPGSERSY DLTGLKPGTEYLVTIYGVKGGDYSPPLSAISTT ISOP193HR9P1G4 SEQ ID NO: 220 LPAPKNLVVSRVTEDSARLSWTAPDAAFDSFEIYYFELRLGGEAIVLTVPGSERSY DLTGLKPGTEYLVTIYGVKGGDYSPPLSAIFTT ISOP193HR9P1G5 SEQ ID NO: 221 LPAPKNLVVSRVTEDSARLSWTAPDAAFDSFDIYYYELRLGGEAIVLTVPGSERSY DLTGLKPGTEYLVTIYGVKGCAASDPLSAIFTT ISOP193HR9P1G6 SEQ ID NO: 222 LPAPKNLVVSRVTEDSARLSWTAPDAAFDSFDIYYYELRLGGEAIVLTVPGSERSY DLTGLKPGTEYLVTIYGVKVCEQSYPLSAISTT ISOP193HR9P1H10 SEQ ID NO: 223 LPAPKNLVVSRVTEDSARLSWTAPDAAFDSFDIYYYELRLGGEAIVLTVPGSERSY DLTGLKPGTEYLVTIYGVKVCLGSNPLSAIFTT ISOP193HR9P1H7 SEQ ID NO: 224 LPAPKNLVVSRVTEDSARLSWTAPDAAFDSFEIYYREPHYGGEAIVLTVPGSERSY DLTGLKPGTEYWVTIYGVKVCLGSNPLSAIFTT 

What is claimed is:
 1. A protein comprising an amino acid sequence that is at least 90% identical to a sequence selected from the group consisting of SEQ ID NOs: 45, 46, 47, 48, 49, 50, 51, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222, 223, and
 224. 2. The protein of claim 1, wherein the amino acid sequence is at least 95% identical to a sequence selected from the group consisting of SEQ ID NOs: 45, 46, 47, 48, 49, 50, 51, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222, 223, and
 224. 3. The protein of claim 1, wherein the protein has at least one substitution at a residue selected from the group consisting of 11, 14, 17, 37, 46, 73, and 86 corresponding to SEQ ID NO:
 4. 4. The protein of claim 1, wherein the protein is conjugated to a heterologous molecule.
 5. The protein of claim 4, wherein the heterologous molecule is a detectable label, a cytotoxic agent, or both.
 6. The protein of claim 5, wherein the cytotoxic agent is a selected from a chemotherapeutic agent, a drug, a growth inhibitory agent, a toxin, an enzymatically active toxin of bacterial origin or fragments thereof, an enzymatically active toxin of fungal origin or fragments thereof, an enzymatically active toxin of plant origin or fragments thereof, an enzymatically active toxin of animal origin or fragments thereof, or a radioactive isotope.
 7. The protein of claim 5, wherein the cytotoxic agent is selected from daunomycin, doxorubicin, methotrexate, vindesine, diphtheria toxin, ricin, geldanamycin, maytansinoids or calicheamicin.
 8. The protein of claim 5, wherein the cytotoxic agent is selected from diphtheria A chain, nonbinding active fragments of diphtheria toxin, exotoxin A chain, ricin A chain, abrin A chain, modeccin A chain, alpha-sarcin, Aleurites fordii proteins, dianthin proteins, Phytolaca americana proteins, Momordica charantia inhibitor, curcin, crotin, Sapaonaria officinalis inhibitor, gelonin, mitogellin, restrictocin, phenomycin, enomycin, or tricothecenes.
 9. The protein of claim 5, wherein the cytotoxic agent is a radionuclide, selected from ²¹²Bi, ¹³¹I, ¹³¹In, ⁹⁰Y, or ¹⁸⁶Re.
 10. The protein of claim 5, wherein the cytotoxic agent is conjugated to the protein by a linker.
 11. The protein of claim 5, wherein the cytotoxic agent is complexed with a chelating agent.
 12. The protein of claim 5, wherein the detectable label is selected from a radioactive isotope, a magnetic bead, a metallic bead, a colloidal particle, a fluorescent dye, an electron-dense reagent, an enzyme, a biotin, a digoxigenin, a hapten, a luminescent molecule, a chemiluminescent molecule, a fluorochrome, a fluorophore, a fluorescent quenching agent, a colored molecule, a cintillant, an avidin, astreptavidin, a protein A, a protein G, an antibody, an antibody fragment, a polyhistidine, a Ni²⁺, a flag tag, a myc tag, a heavy metal, an alkaline phosphatase, a peroxidase, a luciferase, an electron donor, an electron acceptor, an acridinium ester, or a colorimetric substrate.
 13. The protein of claim 5, wherein the detectable label is auristatin, monomethyl auristatin phenylalanine, dolostatin, chemotherapeutic agent, a drug, a growth inhibitory agent, a toxin, or a radioactive isotope.
 14. The protein of claim 5, wherein the detectable label is conjugated to the protein by a linker.
 15. The protein of claim 5, wherein the detectable label is complexed with a chelating agent.
 16. The protein of claim 1, further comprising a methionine at the N-terminus of the protein.
 17. The protein of claim 1, wherein the protein is coupled to a half-life extending moiety.
 18. The protein of claim 17, wherein the half-life extending moiety is an albumin binding molecule, a polyethylene glycol (PEG), albumin, albumin variant, or at least a portion of an Fc region of an immunoglobulin.
 19. A composition comprising the protein of claim 1 and a pharmaceutically acceptable carrier.
 20. A kit comprising the protein of claim
 1. 